Human targets

ABSTRACT

The invention relates to human targets of interest (TOI), anti-TOI ligands, kits compositions and method.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/690,548, filed Nov. 21, 2019, which is a continuation of U.S. patentapplication Ser. No. 14/552,816, filed Nov. 25, 2014, now U.S. Pat. No.10,611,849, which is a continuation of U.S. patent application Ser. No.14/228,760, filed Mar. 28, 2014, now U.S. Pat. No. 8,951,523, which is acontinuation of U.S. patent application Ser. No. 14/138,446, filed Dec.23, 2013, now U.S. Pat. No. 8,883,157, which claims priority to U.S.Provisional Patent Application Ser. No. 61/916,862, filed Dec. 17, 2013,the contents of which are incorporated herein in their entirety byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Dec. 15, 2022, isnamed 732004_SA9-627DIVCON5_SL.xml and is 96,162 bytes in size.

TECHNICAL FIELD

The technology described herein relates to ligands, e.g., antibodies forthe treatment of disease.

BACKGROUND

It is recognized that individual humans differ in their sequence andrecently several individuals have had their genomes sequenced, forinstance James Watson and Craig Venter. Comparison of the genomesequence of individuals has revealed differences in their sequences inboth coding and non-coding parts of the genome. Some of these variationsin humans are significant and contribute to phenotypic differencesbetween individuals. In extreme cases these will result in geneticdisease. The 1000 Genomes Project has the objective of cataloguingsequences in the human genome, involving sequencing the genomes of avery large sampling of individuals from diverse art-recognized humanethnic populations.

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a serineprotease involved in regulating the levels of the low densitylipoprotein receptor (LDLR) protein (Horton et al., 2007; Seidah andPrat, 2007). In vitro experiments have shown that adding PCSK9 to HepG2cells lowers the levels of cell surface LDLR (Benjannet et al., 2004;Lagace et al., 2006; Maxwell et al., 2005; Park et al., 2004).Experiments with mice have shown that increasing PCSK9 protein levelsdecreases levels of LDLR protein in the liver (Benjannet et al., 2004;Lagace et al., 2006; Maxwell et al., 2005; Park et al., 2004), whilePCSK9 knockout mice have increased levels of LDLR in the liver (Rashidet al., 2005). Additionally, various human PCSK9 mutations that resultin either increased or decreased levels of plasma LDL have beenidentified (Kotowski et al., 2006; Zhao et al., 2006). PCSK9 has beenshown to directly interact with the LDLR protein, be endocytosed alongwith the LDLR, and co-immunofluoresce with the LDLR throughout theendosomal pathway (Lagace et al., 2006).

PCSK9 is a prohormone-proprotein convertase in the subtilisin (S8)family of serine proteases (Seidah et al., 2003). Humans have nineprohormone-proprotein convertases that can be divided between the S8Aand S8B subfamilies (Rawlings et al., 2006). Furin, PC1/PC3, PC2, PACE4,PC4, PC5/PC6 and PC7/PC8/LPC/SPC7 are classified in subfamily S8B.Crystal and NMR structures of different domains from mouse furin and PC1reveal subtilisin-like pro- and catalytic domains, and a P domaindirectly C-terminal to the catalytic domain (Henrich et al., 2003;Tangrea et al., 2002). Based on the amino acid sequence similaritywithin this subfamily, all seven members are predicted to have similarstructures (Henrich et al., 2005). SKI-1/SIP and PCSK9 are classified insubfamily S8A. Sequence comparisons with these proteins also suggest thepresence of subtilisin-like pro- and catalytic domains (Sakai et al.,1998; Seidah et al., 2003; Seidah et al., 1999). In these proteins theamino acid sequence C-terminal to the catalytic domain is more variableand does not suggest the presence of a P domain.

Prohormone-proprotein convertases are expressed as zymogens and theymature through a multi step process. The function of the pro-domain inthis process is two-fold. The pro-domain first acts as a chaperone andis required for proper folding of the catalytic domain (Ikemura et al.,1987). Once the catalytic domain is folded, autocatalysis occurs betweenthe pro-domain and catalytic domain. Following this initial cleavagereaction, the pro-domain remains bound to the catalytic domain where itthen acts as an inhibitor of catalytic activity (Fu et al., 2000). Whenconditions are correct, maturation proceeds with a second autocatalyticevent at a site within the pro-domain (Anderson et al., 1997). Afterthis second cleavage event occurs the pro-domain and catalytic domaindissociate, giving rise to an active protease.

Autocatalysis of the PCSK9 zymogen occurs between Gln152 and Ser153(VFAQ|SIP) (Naureckiene et al., 2003), and has been shown to be requiredfor its secretion from cells (Seidah et al., 2003). A secondautocatalytic event at a site within PCSK9's pro-domain has not beenobserved. Purified PCSK9 is made up of two species that can be separatedby non-reducing SDS-PAGE; the pro-domain at 17 Kd, and the catalyticplus C-terminal domains at 65 Kd. PCSK9 has not been isolated withoutits inhibitory pro-domain, and measurements of PCSK9's catalyticactivity have been variable (Naureckiene et al., 2003; Seidah et al.,2003).

In certain embodiments, a PCSK9 polypeptide includes terminal residues,such as, but not limited to, leader sequence residues, targetingresidues, amino terminal methionine residues, lysine residues, tagresidues and/or fusion protein residues. “PCSK9” has also been referredto as FH3, NARC1, HCHOLA3, proprotein convertase subtilisin/kexin type9, and neural apoptosis regulated convertase 1. The PCSK9 gene encodes aproprotein convertase protein that belongs to the proteinase K subfamilyof the secretory subtilase family. The term “PCSK9” denotes both theproprotein and the product generated following autocatalysis of theproprotein. When only the autocatalyzed product is being referred to(such as for an antigen binding protein or ligand that binds to thecleaved PCSK9), the protein can be referred to as the “mature,”“cleaved”, “processed” or “active” PCSK9. When only the inactive form isbeing referred to, the protein can be referred to as the “inactive”,“pro-form”, or “unprocessed” form of PCSK9. The term PCSK9 alsoencompasses PCSK9 molecules incorporating post-translationalmodifications of the PCSK9 amino acid sequence, such as PCSK9 sequencesthat have been glycosylated, PCSK9 sequences from which its signalsequence has been cleaved, PCSK9 sequence from which its pro domain hasbeen cleaved from the catalytic domain but not separated from thecatalytic domain (see, e.g., FIGS. 1A and 1B of US20120093818A1).

SUMMARY

Through the application of human genetic variation analysis andrationally-designed sequence selection the present invention providesfor improved human patient diagnosis and therapy based on human PCSK9variation. Importantly, the invention enables tailored medicines thataddress individual human patient genotypes or phenotypes.

The inventor's analysis of large numbers of naturally-occurring genomichuman PCSK9 sequences reveals that there is significant variation acrossdiverse human populations and provides for the ability for correlationbetween individual human patients and tailored medical and diagnosticapproaches addressing the target. The technical applications of thesefindings, as per the present invention, thus contribute to bettertreatment, prophylaxis and diagnosis in humans and provides for patientbenefit by enabling personalized medicines and therapies. This providesadvantages of better prescribing, less wastage of medications andimproved chances of drug efficacy and better diagnosis in patients.

Furthermore, the inventor surprisingly realised that some rarer naturalforms, although present in humans at much lower frequencies than thecommon form, nevertheless are represented in multiple andethnically-diverse human populations and usually with many humanexamples per represented ethnic population. Thus, the inventor realisedthat targeting such rarer forms would provide for effective treatment,prophylaxis or diagnosis across many human ethnic populations, therebyextending the utility of the present invention and better servingpatients in those populations.

With this, the inventor realised that there is significant industrialand medical application for the invention in terms of guiding the choiceof an anti-PCSK9 ligand for administration to human patients for therapyand/or prophylaxis of PCSK9-mediated or associated diseases andconditions. In this way, the patient receives drugs and ligands that aretailored to their needs—as determined by the patient's genetic orphenotypic makeup. Hand-in-hand with this, the invention provides forthe genotyping and/or phenotyping of patients in connection with suchtreatment, thereby allowing a proper match of drug to patient. Thisincreases the chances of medical efficacy, reduces the likelihood ofinferior treatment using drugs or ligands that are not matched to thepatient (eg, poor efficacy and/or side-effects) and avoidspharmaceutical mis-prescription and waste.

To this end, the invention provides:—

In a First Configuration

An anti-human PCSK9 ligand for use in a method of treating and/orpreventing a PCSK9-mediated disease or condition in a human whose genomecomprises a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 29-37, wherein the method comprises administering the ligandto the human.

In a Second Configuration

A ligand that binds a human PCSK9 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 4-27 for use in amethod comprising the step of using the ligand to target said PCSK9 in ahuman to treat and/or prevent a disease or condition mediated by PCSK9,the method comprising administering the ligand to the human.

In a Third Configuration

A pharmaceutical composition or kit for treating and/or preventing aPCSK9-mediated condition or disease.

In a Fourth Configuration

A method of producing an anti-human PCSK9 antibody binding site, themethod comprising obtaining a plurality of anti-PCSK9 antibody bindingsites, screening the antibody binding sites for binding to a human PCSK9selected from the group consisting of forms f, c, r, p, m, e, h, aj andq or a catalytic or C-terminal domain or a peptide thereof thatcomprises amino acid variation from the corresponding sequence of SEQ IDNO: 1, 2 or 3 and isolating an antibody binding site that binds in thescreening step, and optionally producing a form f, c, r, p, m, e, h, ajor q PCSK9-binding fragment or derivative of the isolated antibody.

In a Fifth Configuration

A method of producing an anti-human PCSK9 antibody, the methodcomprising immunising a non-human vertebrate (eg, a mouse or a rat) witha human PCSK9 comprising an amino acid sequence selected from the groupconsisting of the amino acid sequences of forms f, c, r, p, m, e, h, ajand q or a catalytic or C-terminal domain or a peptide thereof thatcomprises amino acid variation from the corresponding sequence of SEQ IDNO: 1, 2 or 3 and isolating an antibody that binds a human PCSK9comprising selected from the group consisting of forms f, c, r, p, m, e,h, aj and q or a catalytic or C-terminal domain or a peptide thereofthat comprises amino acid variation from the corresponding sequence ofSEQ ID NO: 1, 2 or 3, and optionally producing a form f, c, r, p, m, e,h, aj or q PCSK9-binding fragment or derivative of the isolatedantibody.

In a Sixth Configuration

A kit for PCSK9 genotyping a human, wherein the kit comprises a nucleicacid (i) comprising a sequence of contiguous nucleotides thatspecifically hybridises to a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or at least the catalytic domain- orC-terminal domain-encoding sequence thereof, or specifically hybridisesto an antisense sequence or an RNA transcript of said sequence, whereinsaid sequence of contiguous nucleotides hybridises to at least onenucleotide present in said selected sequence which is not present in SEQID NO: 28 or hybridises to an antisense sequence or an RNA transcriptthereof; and/or (ii) comprising a sequence of at least 10 contiguousnucleotides of a nucleotide sequence selected from the group consistingof SEQ ID NOs: 29-37 or comprising an antisense sequence or RNA versionof said contiguous nucleotides, wherein said sequence of contiguousnucleotides comprises at least one nucleotide present in said selectedsequence which is not present in SEQ ID NO: 28.

In a Seventh Configuration

Use of an anti-PCSK9 ligand that binds a human PCSK9 selected from thegroup consisting of forms f, c, r, p, m, e, h, aj and q in themanufacture of a medicament for treating and/or preventing aPCSK9-mediated disease or condition in a human whose genome comprises anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37.

In a Eighth Configuration

Use of an anti-PCSK9 ligand that binds a human PCSK9 selected from thegroup consisting of forms f, c, r, p, m, e, h, aj and q in themanufacture of a medicament for targeting said PCSK9 in a human to treatand/or prevent a disease or condition mediated by PCSK9.

In a Ninth Configuration

A method of targeting a PCSK9 for treating and/or preventing aPCSK9-mediated disease or condition in a human, the method comprisingadministering an anti-PCSK9 ligand to a human comprising a nucleotidesequence selected from the group consisting SEQ ID NOs: 29-37, whereby aPCSK9 encoded by said nucleotide sequence is targeted.

In a Tenth Configuration

A method of treating and/or preventing a disease or condition mediatedby PCSK9 in a human, the method comprising targeting a human PCSK9selected from the group consisting of forms f, c, r, p, m, e, h, aj andq by administering to the human a ligand that binds said PCSK9 therebytreating and/or preventing said disease or condition in the human.

In a Eleventh Configuration

A method of PCSK9 genotyping a nucleic acid sample of a human, themethod comprising identifying in the sample the presence of a nucleotidesequence selected from the group consisting of SEQ ID NOs: 29-37 or thecatalytic- or C-terminal domain-encoding sequence thereof.

In a Twelfth Configuration

A method of PCSK9 typing a protein sample of a human, the methodcomprising identifying in the sample the presence of a human PCSK9selected from the group consisting of forms f, c, r, p, m, e, h, aj andq.

In a Thirteenth Configuration

A method of treating and/or preventing in a human patient acardiovascular disease or condition, or a disease or condition that isassociated with elevated LDL cholesterol (eg, hypercholesterolaemia),wherein the patient is receiving or has previously received statintreatment for said disease or condition, the method comprising typingthe patient using a method of the invention and administering a ligandaccording to the invention whereby the human is treated or said diseaseor condition is prevented; optionally also reducing or stopping statintreatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows in silico modeling of PCSK9 surface variant residues.

FIG. 2 depicts the cumulative allele frequency distribution across the1000 Genomes Project database of human VH3-23 alleles comprising SNPrs56069819 (such alleles denoted “C” and the most frequent allele (whichdoes not comprise this SNP) denoted “A”).

FIG. 3 depicts frameworks and CDRs encoded by VH3-23*04 as obtained fromthe IMGT database (available on the World Wide Web at www.IMGT.org).Figure discloses SEQ ID NOS 44, 42, 44, 42, 44, 42, 44, 42, 44, 42, 44,42, 44, 42, 44, 42, 44, 42, 45, 43, 44, 42, 47, 46, 47, 46, 47, 46, 47,46, 47, 46, 47, 46, 47, 46, 47, 46, 47, 46, 47, 46, 47, 46, 51, 48, 51,48, 51, 48, 51, 48, 51, 48, 51, 48, 51, 48, 52, 49, 52, 49, 51, 48, 53,50, 57, 54, 57, 54, 57, 54, 57, 54, 57, 54, 57, 54, 58, 55, 59, 56, 59,56, 57, 54, 59, 56, 65, 60, 65, 60, 65, 60, 65, 61, 65, 61, 65, 61, 65,62, 65, 63, 65, 64, 65, 60, 65, and 61, respectively, in order ofappearance.

FIG. 4 depicts sequences of VH3-23*04. The portion of VH3-23*04comprising the FW1 residue change of rs56069819 (SEQ ID NO: 38). Theportion of the nucleic acid sequence encoding rs56069819 is depicted(SEQ ID NO: 39). The FW1 encoded by VH3-23*04 is depicted (SEQ ID NO:40).

DETAILED DESCRIPTION

The skilled person will know that SNPs or other changes that translateinto amino acid variation can cause variability in conformation oractivity of human targets to be addressed. This has spawned greatinterest in personalized medicine where genotyping and knowledge ofprotein and nucleotide variability is used to tailor medicines anddiagnosis of patients more effectively. The present invention providesfor tailored pharmaceuticals and testing that specifically addressesrarer variant forms of a human target of interest (TOI), that targetbeing human PCSK9.

The present invention harnesses the power of human genetic variationanalysis and rationally-designed sequence selection. The technicalapplications of these approaches, as per the present invention,contribute to better treatment, prophylaxis and diagnosis in humans andprovides for patient benefit by providing choice and enablingpersonalized medicines and therapies. This provides advantages of betterprescribing, less wastage of medications and improved chances of drugefficacy and better diagnosis in patients.

As sources of genomic sequence variation data, the skilled person willbe aware of the available databases and resources (including updatesthereof) provided by the following:—

-   1. HapMap (The International HapMap Consortium. 2003;    http://hapmap.ncbi.nlm.nih.gov/index.html.en). The HapMap Project is    an international project that aims to compare the genetic sequences    of different individuals to identify chromosomal regions containing    shared genetic variants. The HapMap www site provides tools to    identify chromosomal regions and the variant therein, with options    to drill down to population level frequency data.-   2. 1000 Genomes Project (The 1000 Genomes Project Consortium 2010;    available on the World Wide Web at http://www.1000genomes.org/).    This resource provides complete genomic sequence for at least 2500    unidentified individuals from one of 25 distinct population groups.-   3. Japanese SNP Database (H. Haga et al. 2002; available on the    World Wide Web at http://snp.ims.u-tokyo.ac.jp/index.html). Based on    a study identifying 190,562 human genetic variants.

The present invention involves the identification and cataloguing ofnaturally-occurring human genomic target sequence variants, includingthose found to be relatively low-frequency or rare variants thatsegregate with specific human ethnic populations and in many individualhumans.

An aspect of the invention is based on rational design of sequenceselection addressing the desirability to tailor medicaments anddiagnostics to rarer, but yet still significant groups of humanindividuals that suffer from, or have the potential to suffer from (ie,who are at risk of), a disease or condition mediated or associated withthe target of interest. In devising this rational design of the presentaspect of the invention, the inventor included considerations of thespread of prevalence of naturally-occurring target variant sequencesacross multiple, diverse human ethnic populations, as well as theimportance of addressing such populations where many individuals arelikely to display a genotype and/or phenotype of one or more of thevariants being analysed. As part of this design, the inventor saw theimportance of adopting the art-recognised classifications of humanethnic populations, and in this respect the inventor based the analysisand design on the recognised human ethnic populations adopted by the1000 Genomes Project, since this is a resource that is, and willcontinue to be, widely adopted by the scientific and medical community.

FIG. 2 shows the cumulative allele frequency distribution across the1000 Genomes Project database of human VH3-23 alleles comprising SNPrs56069819 (such alleles denoted “C” and the most frequent allele (whichdoes not comprise this SNP) denoted “A”). The figure shows that VH3-23alleles comprising SNP rs56069819 are present at a cumulative frequencyof 11% across all human ethnic populations taken as a whole, whereas incertain specific human ethnic sub-populations (ASW, LWK, YRI, CEU andGBR) such alleles are present at an above-average cumulative frequency.Indicated in the figure are those human PCSK9 variant forms (marked“Variants”) that are found in the various sub-populations withabove-average occurrence of human VH3-23 alleles comprising SNPrs56069819.

Thus, in this aspect of the invention, the inventor designed thefollowing variant sequence selection criteria, these being criteria thatthe inventor realised would provide for useful medical drugs anddiagnostics to tailored need in the human population.

Selection Criteria

Three or four of the following:—

-   -   Naturally-occurring human PCSK9 variant sequences having a        cumulative human allele frequency of 35% or less;    -   Naturally-occurring human PCSK9 variant sequences having a total        human genotype frequency of 40% or less;    -   Naturally-occurring human PCSK9 variant sequences found in many        different human ethnic populations (using the standard        categorisation of the 1000 Genomes Project; see Table 3 below);        and    -   Naturally-occurring human PCSK9 variant sequences found in many        individuals distributed across such many different ethnic        populations.

The inventor's selection included, as a consideration, selection fornucleotide variation that produced amino acid variation in correspondingPCSK9 forms (ie, non-synonymous variations), as opposed to silentvariations that do not alter amino acid residues in the target protein.

Optionally, further sequence analysis and 3D in silico modelling (eg,see FIG. 1 ) can also be used as an additional selection criterion:variants whose variant amino acid residues (versus the most common formof human PCSK9) are surface-exposed on the target are desirable forselection, since the inventor saw these as contributing to determiningthe topography of the target and potentially contributing to how andwhere ligand binding on the target occurs.

In an embodiment, the cumulative human allele frequency is 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15% or 1 to10%.

In an embodiment, the total human genotype frequency is 35, 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%, 1 to15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 to about5%.

In an embodiment, the naturally-occurring human target variant sequencesare found in at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20different human ethnic populations (using the standard categorisation ofthe 1000 Genomes Project).

In an embodiment, the naturally-occurring human target variant sequencesare found in at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150 individualsdistributed across such many different ethnic populations.

In an example, the following criteria are applied:—

-   -   Naturally-occurring human PCSK9 variant sequences having a        cumulative human allele frequency of 15% or less;    -   Naturally-occurring human PCSK9 variant sequences having a total        human genotype frequency of 20% or less;    -   Naturally-occurring human PCSK9 variant sequences found in at        least 5 different human ethnic populations (using the standard        categorisation of the 1000 Genomes Project); and    -   Naturally-occurring human PCSK9 variant sequences found in many        individuals distributed across such many different ethnic        populations.

In any aspect, configuration, example, embodiment, clause or conceptherein, frequencies may be determined using bioinformatics.

In any aspect, configuration, example, embodiment, clause or conceptherein, frequencies may be determined by reference to a databasecomprising at least 1000 or 2000 human sequences.

In any aspect, configuration, example, embodiment, clause or conceptherein “heterozygous human genotype frequency” means the cumulativefrequency of all genotypes in the sample or database or in humans havingone occurrence of the rare variant allele and one occurrence of anotherallele (heterozygous state), eg, genotype in 1000 Genomes database.

In any aspect, configuration, example, embodiment, clause or conceptherein “homozygous human genotype frequency” means the cumulativefrequency of two occurrences of the variant allele (homozygous state),eg, genotype in 1000 Genomes Project database.

In any aspect, configuration, example, embodiment, clause or conceptherein “total human genotype frequency” means the total of heterozygousplus homozygous human genotype frequencies.

In any aspect, configuration, example, embodiment, clause or conceptherein “cumulative human allele frequency” refers to the total of alloccurrences of the variant allele in the sample or database or inhumans, eg, in the 1000 Genomes Project database.

In an example, the criteria are applied with reference to one or morehuman genomic sequence databases as described herein. For example, thecriteria are those as applied to the 1000 Genomes database.

For example in any aspect example, embodiment or configuration of theinvention, the 1000 Genomes database release 13. For example, the 1000Genomes database in its most recent version as at 1 Oct. 2013.

The following bioinformatics protocol is envisaged to identify humansequences for use in the present invention:

-   -   (a) Identify a genomic region containing a PCSK9 target sequence        of interest (‘target genomic region’) and calculate the genomic        coordinates, using coordinates that match the sequence assembly        build used by either the 1000 Genomes Project or International        HapMap project (or another selected human gene database of        choice).    -   (b) Identify genomic variants mapped to the genomic region        previously identified in (a). Retrieve allele frequencies for        variants for each super population and preferably sub-population        where such data is available. The VWC tools for the 1000 Genomes        Project can be used for this step.    -   (c) Filter list of genomic variants from target genomic region        to contain only variants classed as either ‘non-synonymous’        single nucleotide polymorphisms (SNPs) or genomic ‘insertions or        delections’ (indels). Filter further to include those that are        present in exonic sequences only. “Non-synonymous” refers to        nucleotide variation that produces amino acid variation (ie,        excluding silent mutations).    -   (d) Correlate population frequency data for each of the        identified variants for each of the super populations (for        example ‘European Ancestry’, ‘East Asian ancestry’, ‘West        African ancestry’, ‘Americas’, and ‘South Asian ancestry’) to        identify those variants that segregate with less than two        super-populations. Further correlate all identified variants        with each of the sub-populations (for example, ‘European        ancestry’ super-population might be subdivided into groups such        as ‘CEU—Utah residents with Northern or Western European        ancestry’, ‘TSI Toscani in Italia’ and ‘British from England and        Scotland’) and produce a second score for rarity of variants        within a super-population.    -   (e) Collect one or more sequences that show segregation to        specific sub-populations for use in the present invention, eg,        according to selection criteria as described herein.

Human Populations

Optionally the ethnic populations are selected from those identified inthe 1000 Genomes Project database. In this respect, see Table 3 whichprovides details of the ethnic populations on which the 1000 GenomesProject database is based.

N A Rosenberg et al (Science 20 Dec. 2002: vol. 298 no. 5602 2342-2343)studied the genetic structure of human populations of differinggeographical ancestry. In total, 52 populations were sampled, thesebeing populations with:

African Ancestry

(Mbuti Pygmies, Biaka Pygmies, San peoples, and speakers ofNiger-Kordofanian languages (Bantu, Yoruba or Mandenka populations),

Eurasian Ancestry

(European ancestry (Orcadian, Adygel, Basque, French, Russians,Italians, Sardinian, Tuscan), Middle Eastern ancestry (Mozabite,Bedouin, Druze, Palestinians), Central/South Asian ancestry (Balochl,Brahul, Makrani, Sindhi, Pathan, Burusho, Hazara, Uygur, Kalash)),

East Asian Ancestry

(Han, Dal, Daur, Hezhen, Lahu, Miao, Oroqen, She, Tujia, Tu, Xibo, Yi,Mongola, Naxi, Cambodian, Japanese, Yakut), Oceanic ancestry(Melanesian, Papuan); or

Americas Ancestry

(Karitiana, Surui, Colombian, Maya, Pima).

The International HapMap Project, Nature, 2003 Dec. 18;426(6968):789-96, discloses that goal of the HapMap Project: todetermine the common patterns of DNA sequence variation in the humangenome by determining the genotypes of one million or more sequencevariants, their frequencies and the degree of association between themin DNA samples from populations with ancestry from parts of Africa, Asiaand Europe. The relevant human populations of differing geographicalancestry include Yoruba, Japanese, Chinese, Northern European andWestern European populations. More specifically:—

Utah population with Northern or Western European ancestry (samplescollected in 1980 by the Centre d'Etude du Polymorphisme Humain (CEPH));

population with ancestry of Yoruba people from Ibadan, Nigeria;population with Japanese ancestry; andpopulation with ancestry of Han Chinese from China.

The authors, citing earlier publications, suggest that ancestralgeography is a reasonable basis for sampling human populations.

A suitable sample of human populations used in the present invention isas follows:—

-   -   (a) European ancestry    -   (b) Northern European ancestry; Western European ancestry;        Toscani ancestry; British ancestry, Finnish ancestry or Iberian        ancestry.    -   (c) More specifically, population of Utah residents with        Northern and/or Western European ancestry; Toscani population in        Italia; British population in England and/or Scotland; Finnish        population in Finland; or Iberian population in Spain.    -   (a) East Asian ancestry    -   (b) Japanese ancestry; Chinese ancestry or Vietnamese ancestry.    -   (c) More specifically, Japanese population in Toyko, Japan; Han        Chinese population in Beijing, China; Chinese Dai population in        Xishuangbanna; Kinh population in Ho Chi Minh City, Vietnam; or        Chinese population in Denver, Colo., USA.    -   (a) West African ancestry    -   (b) Yoruba ancestry; Luhya ancestry; Gambian ancestry; or        Malawian ancestry.    -   (c) More specifically, Yoruba population in Ibadan, Nigeria;        Luhya population in Webuye, Kenya; Gambian population in Western        Division, The Gambia; or Malawian population in Blantyre,        Malawi.    -   (a) Population of The Americas    -   (b) Native American ancestry; Afro-Caribbean ancestry; Mexican        ancestry; Puerto Rican ancestry; Columbian ancestry; or Peruvian        ancestry.    -   (c) More specifically, population of African Ancestry in        Southwest US; population of African American in Jackson, Miss.;        population of African Caribbean in Barbados; population of        Mexican Ancestry in Los Angeles, Calif.; population of Puerto        Rican in Puerto Rico; population of Colombian in Medellin,        Colombia; or population of Peruvian in Lima, Peru.    -   (a) South Asian ancestry    -   (b) Ahom ancestry; Kayadtha ancestry; Reddy ancestry; Maratha;        or Punjabi ancestry.    -   (c) More specifically, Ahom population in the State of Assam,        India; Kayadtha population in Calcutta, India; Reddy population        in Hyderabad, India; Maratha population in Bombay, India; or        Punjabi population in Lahore, Pakistan.

In any configuration of the invention, in one embodiment, each humanpopulation is selected from a population marked “(a)” above.

In any configuration of the invention, in another embodiment, each humanpopulation is selected from a population marked “(b)” above.

In any configuration of the invention, in another embodiment, each humanpopulation is selected from a population marked “(c)” above.

In one embodiment the ethnic populations are selected from the groupconsisting of an ethnic population with European ancestry, an ethnicpopulation with East Asian, an ethnic population with West Africanancestry, an ethnic population with Americas ancestry and an ethnicpopulation with South Asian ancestry.

In one embodiment the ethnic populations are selected from the groupconsisting of an ethnic population with Northern European ancestry; oran ethnic population with Western European ancestry; or an ethnicpopulation with Toscani ancestry; or an ethnic population with Britishancestry; or an ethnic population with Icelandic ancestry; or an ethnicpopulation with Finnish ancestry; or an ethnic population with Iberianancestry; or an ethnic population with Japanese ancestry; or an ethnicpopulation with Chinese ancestry; or an ethnic population Vietnameseancestry; or an ethnic population with Yoruba ancestry; or an ethnicpopulation with Luhya ancestry; or an ethnic population with Gambianancestry; or an ethnic population with Malawian ancestry; or an ethnicpopulation with Native American ancestry; or an ethnic population withAfro-Caribbean ancestry; or an ethnic population with Mexican ancestry;or an ethnic population with Puerto Rican ancestry; or an ethnicpopulation with Columbian ancestry; or an ethnic population withPeruvian ancestry; or an ethnic population with Ahom ancestry; or anethnic population with Kayadtha ancestry; or an ethnic population withReddy ancestry; or an ethnic population with Maratha; or an ethnicpopulation with Punjabi ancestry.

Anti-Target Ligands

The invention provides useful anti-target ligands for addressing humanssuffering from or likely to suffer from a disease or condition mediatedor associated with PCSK9. For example, the ligand specifically binds toa PCSK9 variant as per the invention. The ligand may inhibit orantagonise the activity of the PCSK9 target, eg, the ligand neutralisesthe target. The skilled person will be familiar with neutralisingligands in general, such as antibodies or antibody fragments, and canreadily test suitable ligands for specific binding and/or neutralisationof a target in vitro or in an in vivo assay.

In an example, the ligand is (or has been determined as) a neutraliserof the PCSK9. In an example, determination is carried out in a human(eg, in a clinical trial). In an example, determination is carried outin a non-human, eg, in a mouse, rat, rabbit, pig, dog, sheep ornon-human primate (eg, Cynomolgous monkey, rhesus monkey or baboon).

An antibody “fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include dAb, Fab, Fab′, F(ab′)2and Fv fragments; diabodies; linear antibodies; single-chain antibodymolecules and multispecific antibodies formed from antibody fragments.

In an embodiment, the ligand of the invention is or comprises anantibody or antibody fragment, for example an antibody or fragmentcomprising human variable regions (and optionally also human constantregions). Anti-PCSK9 or PCSK9-binding or targeting antibodies andfragments can be prepared according to any known method, eg, usingtransgenic mice (eg, the Kymouse™ or Velocimouse™, or Omnimouse™,Xenomouse™, HuMab Mouse™ or MeMo Mouse™), rats (eg, the Omnirat™),camelids, sharks, rabbits, chickens or other non-human animals immunisedwith the PCSK9 followed optionally by humanisation of the constantregions and/or variable regions to produce human or humanisedantibodies. In an example, display technologies can be used, such asyeast, phage or ribosome display, as will be apparent to the skilledperson. Standard affinity maturation, eg, using a display technology,can be performed in a further step after isolation of an antibody leadfrom a transgenic animal, phage display library or other library.Representative examples of suitable technologies are described inUS20120093818 (Amgen, Inc), which is incorporated by reference herein inits entirety, eg, the methods set out in paragraphs [0309] to [0346].

Generally, a VELOCIMMUNE™ or other mouse or rat can be challenged withthe antigen of interest, and lymphatic cells (such as B-cells) arerecovered from the mice that express antibodies. The lymphatic cells maybe fused with a myeloma cell line to prepare immortal hybridoma celllines, and such hybridoma cell lines are screened and selected toidentify hybridoma cell lines that produce antibodies specific to theantigen of interest. DNA encoding the variable regions of the heavychain and light chain may be isolated and linked to desirable isotypicconstant regions of the heavy chain and light chain. Such an antibodyprotein may be produced in a cell, such as a CHO cell. Alternatively,DNA encoding the antigen-specific chimaeric antibodies or the variabledomains of the light and heavy chains may be isolated directly fromantigen-specific lymphocytes.

Initially, high affinity chimaeric antibodies are isolated having ahuman variable region and a mouse constant region. As described below,the antibodies are characterized and selected for desirablecharacteristics, including affinity, selectivity, epitope, etc. Themouse constant regions are replaced with a desired human constant regionto generate the fully human antibody of the invention, for examplewild-type or modified IgG1 or IgG4 (for example, SEQ ID NO: 751, 752,753 in US2011/0065902 (which is incorporated by reference herein in itsentirety), which sequences are incorporated herein by reference for usein the ligands of the present invention). While the constant regionselected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region.

In an example, the ligand of the invention is or comprises a nucleicacid, eg, RNA, eg, siRNA that hybridises under stringent condition tothe PCSK9 variant sequence, eg, hybridises a nucleotide sequencecomprising one or more nucleotides that are variant (versus the mostcommon PCSK9 sequence, eg, with reference to the 1000 Genomes Projectdatabase).

For example, the nucleic acid hybridises to a region immediatelyflanking a nucleotide that is variant compared to the correspondingnucleotide of the PCSK9 nucleotide sequence having the highestcumulative human allele frequency and/or the highest total humangenotype frequency. In an example, the nucleic acid hybridises to at twoor more such variant nucleotides.

Specific hybridisation is under stringent conditions, as will beapparent to the skilled person, eg, conditions of 5×SSC, 5×Denhardt'sreagent, and 0.5% SDS at 65° C.

Target binding ability, specificity and affinity (Kd, K_(off) and/orK_(on)) can be determined by any routine method in the art, eg, bysurface plasmon resonance (SPR). The term “Kd”, as used herein, isintended to refer to the equilibrium dissociation constant of aparticular antibody-antigen interaction.

In one embodiment, the surface plasmon resonance (SPR) is carried out at25° C. In another embodiment, the SPR is carried out at 37° C.

In one embodiment, the SPR is carried out at physiological pH, such asabout pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (alsoreferred to as HBS-EP)).

In one embodiment, the SPR is carried out at a physiological salt level,eg, 150 mM NaCl.

In one embodiment, the SPR is carried out at a detergent level of nogreater than 0.05% by volume, eg, in the presence of P20 (polysorbate20; eg, Tween-20™) at 0.05% and EDTA at 3 mM.

In one example, the SPR is carried out at 25° C. or 37° C. in a bufferat pH7.6, 150 mM NaCl, 0.05% detergent (eg, P20) and 3 mM EDTA. Thebuffer can contain 10 mM Hepes. In one example, the SPR is carried outat 25° C. or 37° C. in HBS-EP. HBS-EP is available from Teknova Inc(California; catalogue number H8022).

In an example, the affinity of the ligand (eg, antibody) is determinedusing SPR by

-   1. Coupling anti-mouse (or other relevant human, rat or non-human    vertebrate antibody constant region species-matched) IgG (eg,    Biacore™ BR-1008-38) to a biosensor chip (eg, GLM chip) such as by    primary amine coupling;-   2. Exposing the anti-mouse IgG (or other matched species antibody)    to a test IgG antibody to capture test antibody on the chip;-   3. Passing the test antigen over the chip's capture surface at 1024    nM, 256 nM, 64 nM, 16 nM, 4 nM with a 0 nM (i.e. buffer alone); and-   4. And determining the affinity of binding of test antibody to test    antigen using surface plasmon resonance, eg, under an SPR condition    discussed above (eg, at 25° C. in physiological buffer). SPR can be    carried out using any standard SPR apparatus, such as by Biacore™ or    using the ProteOn XPR36™ (Bio-Rad®).

Regeneration of the capture surface can be carried out with 10 mMglycine at pH1.7. This removes the captured antibody and allows thesurface to be used for another interaction. The binding data can befitted to 1:1 model inherent using standard techniques, eg, using amodel inherent to the ProteOn XPR36™ analysis software.

In an example, the ligand of the invention is contained in a medicalcontainer, eg, a vial, syringe, IV container or an injection device (eg,an intraocular or intravitreal injection device). In an example, theligand is in vitro, eg, in a sterile container. In an example, theinvention provides a kit comprising the ligand of the invention,packaging and instructions for use in treating or preventing ordiagnosing in a human a disease or condition mediated by the PCSK9. Inan example, the instructions indicate that the human should be genotypedfor a PCSK9 variant sequence of the invention before administering theligand to the human. In an example, the instructions indicate that thehuman should be phenotyped for a PCSK9 variant of the invention beforeadministering the ligand to the human. In an example, the human is ofChinese (eg, Han or CHS) ethnicity and the instructions are in Chinese(eg, Mandarin). In an example, the instructions comprise directions toadminister alirocumab or evolocumab to said human.

The invention addresses the need to treat humans havingnaturally-occurring rarer natural PCSK9 alleles, genotypes andphenotypes (rarer protein forms). In this respect, the inventionprovides the following aspects.

In a First Aspect: An anti-human PCSK9 ligand for use in a method oftreating and/or preventing a PCSK9-mediated disease or condition in ahuman whose genome comprises a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 29-37, wherein the method comprisesadministering the ligand to the human.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

The PCSK9 variant is not the most frequent.

In an embodiment of any configuration, example, embodiment or aspectherein, the ligand, antibody, fragment or binding site of the inventionis recombinant.

In a Second Aspect: The ligand of aspect 1, wherein the ligand has beenor is determined as capable of binding a human PCSK9 selected from thegroup consisting forms f c, r, p, m, e, h, aj and q.

In an example of any aspect, the ligand binds (or has been determined tobind) two, three, four or more human PCSK9 selected from the groupconsisting forms f, c, r, p, m, e, h, aj and q.

In an example of any aspect, the ligand comprises a protein domain thatspecifically binds to PCSK9, eg, a human PCSK9 selected from the groupconsisting forms f; c, r, p, m, e, h, aj and q.

The term “specifically binds,” or the like, means that a ligand, eg, anantibody or antigen-binding fragment thereof, forms a complex with anantigen that is relatively stable under physiologic conditions. Specificbinding can be characterized by an equilibrium dissociation constant ofat least about 1×10⁻⁶ M or less (e.g., a smaller KD denotes a tighterbinding). Methods for determining whether two molecules specificallybind are well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like. An isolated antibodythat specifically binds a human PCSK9 may, however, exhibitcross-reactivity to other antigens such as a PCSK9 molecule from anotherspecies. Moreover, multi-specific antibodies (e.g., bispecifics) thatbind to human PCSK9 and one or more additional antigens are nonethelessconsidered antibodies that “specifically bind” PCSK9, as used herein.

In an example of any aspect, the ligand comprises or consists of aprotein that mimics the EGFA domain of the LDL receptor and specificallybinds to PCSK9, eg, a human PCSK9 selected from the group consistingforms f, c, r, p, m, e, h, aj and q.

In an example of any aspect, the ligand antagonises PCSK9, eg, a humanPCSK9 selected from the group consisting forms f, c, r, p, m, e, h, ajand q.

In an example of any aspect, the method comprises (before administeringthe ligand) the step of determining that the ligand is capable ofbinding a human PCSK9 selected from the group consisting forms f, c, r,p, m, e, h, aj and q.

In an example of any aspect, binding is determined by SPR. In an exampleof any aspect, binding is determined by ELISA.

In an example of any aspect, said forms are the mature forms.

In an example of any aspect, said forms are the pro-forms.

The terms “is determined”, “is genotyped” or “is phenotyped” and thelike herein mean that the method comprises a step of such determining,genotyping or phenotyping.

In a Third Aspect: A ligand that binds a human PCSK9 comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 4-27 foruse in a method comprising the step of using the ligand to target saidPCSK9 in a human to treat and/or prevent a disease or condition mediatedby PCSK9, the method comprising administering the ligand to the human.

In an example, the disease or condition is mediated by a human PCSK9comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 4-27.

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4-23, 26 and 27; or selected from the groupconsisting of SEQ ID NOs: 4-14 and 18-27; or selected from the groupconsisting of SEQ ID NOs: 4-14, 18-23, 26 and 27. These arenaturally-occurring sequences that do not comprise 46L and which meetthe criteria set out above. These groups comprise variants that areassociated with elevated LDL-C.

In an example, the amino acid sequence is SEQ ID NO: 18, 19 or 20, thatcomprises a 425S, which is associated with elevated LDL-C (Pisciotta etal 2006).

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 11, 12, 26 and 27, that comprise 670Gwhich is a marker for severity of coronary atherosclerosis (Chen et al2005).

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10-14 and 18-27; or selected from the groupconsisting of SEQ ID NOs: 10-14, 18-23, 26 and 27. These are sequencesthat have a naturally-occurring combination of differences from SEQ IDNOs: 1-3 (form a) and which meet the criteria set out above.

In an example, the amino acid sequence is SEQ ID NO: 4.

In an example, the amino acid sequence is SEQ ID NO: 5.

In an example, the amino acid sequence is SEQ ID NO: 6.

In an example, the amino acid sequence is SEQ ID NO: 7.

In an example, the amino acid sequence is SEQ ID NO: 8.

In an example, the amino acid sequence is SEQ ID NO: 9.

In an example, the amino acid sequence is SEQ ID NO: 10.

In an example, the amino acid sequence is SEQ ID NO: 11.

In an example, the amino acid sequence is SEQ ID NO: 12.

In an example, the amino acid sequence is SEQ ID NO: 13.

In an example, the amino acid sequence is SEQ ID NO: 14.

In an example, the amino acid sequence is SEQ ID NO: 15.

In an example, the amino acid sequence is SEQ ID NO: 16.

In an example, the amino acid sequence is SEQ ID NO: 17.

In an example, the amino acid sequence is SEQ ID NO: 18.

In an example, the amino acid sequence is SEQ ID NO: 19.

In an example, the amino acid sequence is SEQ ID NO: 20.

In an example, the amino acid sequence is SEQ ID NO: 21.

In an example, the amino acid sequence is SEQ ID NO: 22.

In an example, the amino acid sequence is SEQ ID NO: 23.

In an example, the amino acid sequence is SEQ ID NO: 24.

In an example, the amino acid sequence is SEQ ID NO: 25.

In an example, the amino acid sequence is SEQ ID NO: 26.

In an example, the amino acid sequence is SEQ ID NO: 27.

In a Fourth Aspect: The ligand of aspect 3, wherein the genome of thehuman comprises a nucleotide sequence selected from the group consistingof SEQ ID NOs: 29-37.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Fifth Aspect: The ligand of any preceding aspect, wherein the humanhas been or is genotyped as positive for a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 29-37 or at least the catalyticdomain- or C-terminal domain-encoding sequence thereof.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37.

These are naturally-occurring allele (haplotype) sequences that do notencode 46L and which meet the criteria set out above. These groupscomprise variants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Sixth Aspect: The ligand of any preceding aspect, wherein the humanhas been or is phenotyped as positive for a human PCSK9 selected fromthe group consisting of forms f, c, r, p, m, e, h, aj and q or at leastthe catalytic or C-terminal domain thereof.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Seventh Aspect: The ligand of any preceding aspect, wherein themethod comprises genotyping the human as positive for a nucleotidesequence selected from the group consisting of SEQ ID NOs: 29-37 or atleast the catalytic domain- or C-terminal domain-encoding sequencethereof.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In an Eight Aspect: The ligand of any preceding aspect, wherein themethod comprises phenotyping the human has positive for a human PCSK9selected from the group consisting of forms f, c, r, p, m, e, h, aj andq or at least the catalytic or C-terminal domain thereof.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Ninth Aspect: The ligand of any preceding aspect, wherein the humanhas been or is genotyped as heterozygous for a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 29-37 or at least thecatalytic domain- or C-terminal domain-encoding sequence thereof;optionally wherein the human has been or is genotyped as comprising thenucleotide sequence of SEQ ID NO: 28 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof and a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 29-37 or at least thecatalytic domain- or C-terminal domain-encoding sequence thereof.

“Heterozygous” here means that in the human's genotype one allelecomprises a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminaldomain-encoding sequence thereof and other allele can be any PCSK9 (eg,form a, a′ or an allele comprising a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 29-37 or at least the catalyticdomain- or C-terminal domain-encoding sequence thereof).

In an example, the method comprises (before administering the ligand)genotyping the human as heterozygous for a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 29-37 or at least the catalyticdomain- or C-terminal domain-encoding sequence thereof; optionally alsogenotyping the human as comprising the nucleotide sequence of SEQ ID NO:28 or at least the catalytic domain- or C-terminal domain-encodingsequence thereof and a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or at least the catalytic domain- orC-terminal domain-encoding sequence thereof.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Tenth Aspect: The ligand of any one of aspects 1 to 9, wherein thegenome of the human has been or is genotyped as homozygous for anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37 or at least the catalytic domain- or C-terminal domain-encodingsequence thereof.

“Homozygous” here means that in the human's genotype each allelecomprises the same nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or at least the catalytic domain- orC-terminal domain-encoding sequence thereof.

In an example, the method comprises genotyping the human as homozygousfor a nucleotide sequence selected from the group consisting of SEQ IDNOs: 29-37 or at least the catalytic domain- or C-terminaldomain-encoding sequence thereof.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In an Eleventh Aspect: The ligand of any preceding aspect, wherein theligand comprises an antibody binding site that binds a human PCSK9comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 4-27 and optionally has been or is determined as capable ofsuch binding.

In an example, the method comprises (before administering the ligand)the step of determining that the ligand is capable of binding to saidhuman PCSK9.

In an example, the binding is specific binding. In an example, theligand binds (or has been determined as binding) to the PCSK9 with anaffinity (Kd) of 1 mM, 100 nM, 10 nM or 1 nM or less. In an embodiment,the affinity is no less than 10, 100 or 1000 fM.

In an example, binding or affinity is determined by SPR or ELISA.

In an example, the disease or condition is mediated by a human PCSK9comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 4-27.

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4-23, 26 and 27; or selected from the groupconsisting of SEQ ID NOs: 4-14 and 18-27; or selected from the groupconsisting of SEQ ID NOs: 4-14, 18-23, 26 and 27. These arenaturally-occurring sequences that do not comprise 46L and which meetthe criteria set out above. These groups comprise variants that areassociated with elevated LDL-C.

In an example, the amino acid sequence is SEQ ID NO: 18, 19 or 20, thatcomprises a 425S, which is associated with elevated LDL-C (Pisciotta etal 2006).

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 11, 12, 26 and 27, that comprise 670Gwhich is a marker for severity of coronary atherosclerosis (Chen et al2005).

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10-14 and 18-27; or selected from the groupconsisting of SEQ ID NOs: 10-14, 18-23, 26 and 27. These are sequencesthat have a naturally-occurring combination of differences from SEQ IDNOs: 1-3 (form a) and which meet the criteria set out above.

In an example, the amino acid sequence is SEQ ID NO: 4.

In an example, the amino acid sequence is SEQ ID NO: 5.

In an example, the amino acid sequence is SEQ ID NO: 6.

In an example, the amino acid sequence is SEQ ID NO: 7.

In an example, the amino acid sequence is SEQ ID NO: 8.

In an example, the amino acid sequence is SEQ ID NO: 9.

In an example, the amino acid sequence is SEQ ID NO: 10.

In an example, the amino acid sequence is SEQ ID NO: 11.

In an example, the amino acid sequence is SEQ ID NO: 12.

In an example, the amino acid sequence is SEQ ID NO: 13.

In an example, the amino acid sequence is SEQ ID NO: 14.

In an example, the amino acid sequence is SEQ ID NO: 15.

In an example, the amino acid sequence is SEQ ID NO: 16.

In an example, the amino acid sequence is SEQ ID NO: 17.

In an example, the amino acid sequence is SEQ ID NO: 18.

In an example, the amino acid sequence is SEQ ID NO: 19.

In an example, the amino acid sequence is SEQ ID NO: 20.

In an example, the amino acid sequence is SEQ ID NO: 21.

In an example, the amino acid sequence is SEQ ID NO: 22.

In an example, the amino acid sequence is SEQ ID NO: 23.

In an example, the amino acid sequence is SEQ ID NO: 24.

In an example, the amino acid sequence is SEQ ID NO: 25.

In an example, the amino acid sequence is SEQ ID NO: 26.

In an example, the amino acid sequence is SEQ ID NO: 27.

In a Twelfth Aspect: The ligand of aspect 11, wherein the ligand is anantibody or antibody fragment. For example, the antibody or antibodyfragment is a PCSK9 antagonist, eg, neutralises PCSK9. Examples of suchantibodies are disclosed, for instance, in WO 2008/057457,WO2008/057458, WO 2008/057459, WO 2008/063382, WO 2008/133647, WO2009/100297, WO 2009/100318, WO 2011/037791, WO 2011/053759, WO2011/053783, WO 2008/125623, WO 2011/072263, WO 2009/055783, WO2010/029513, WO 2011/11 1007, WO 2010/077854, the disclosures andsequences of such antibodies being incorporated herein in theirentireties by refere for use in the invention. One specific example isAMG 145 (Amgen), LY3015014 (Eli Lilly) or alirocumab, or a PCSK9-bindingderivative thereof. Advantageously, the ligand is or comprisesalirocumab.

Alternatively, the ligand is or comprises evolocumab.

In an example, the ligand is SAR236553/REGN727 (SanofiAventis/Regeneron) or a PCSK9-binding derivative thereof.

In an example, the ligand comprises or consists of a neutralizingantibody that binds to the PCSK9, wherein the antibody binds to PCSK9and reduces the likelihood that PCSK9 binds to LDLR.

The ligand of aspect 11, wherein the ligand is a PCSK9 antagonist, eg,neutralises PCSK9.

In an example of any aspect of the invention, the ligand comprises orconsists a ligand selected from evolocumab, 1D05-IgG2 (Merck & Co.),ALN-PCS02 (Alnylam), RN316 (Pfizer-Rinat), LY3015014 (Eli Lilly) andalirocumab, or a PCSK9-binding derivative thereof. In an example, theligand is SAR236553/REGN727 (Sanofi Aventis/Regeneron) or aPCSK9-binding derivative thereof.

In a Thirteenth Aspect: The ligand of any one of aspects 1 to 10,wherein (i) the ligand comprises a sequence of contiguous nucleotidesthat specifically hybridises to a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 29-37 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof, or specificallyhybridises to an antisense sequence or an RNA transcript of saidsequence, wherein said sequence of contiguous nucleotides hybridises toat least one nucleotide present in said selected sequence which is notpresent in SEQ ID NO: 28 or hybridises to an antisense sequence or anRNA transcript thereof respectively; and/or (ii) the ligand comprises asequence of at least 10 contiguous nucleotides of a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 29-37 or is anantisense sequence or RNA version of said contiguous nucleotides,wherein said sequence of contiguous nucleotides comprises at least onenucleotide present in said selected sequence which is not present in SEQID NO: 28.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In an embodiment, the ligand comprises at least 10, 11, 12, 13, 14, 15,20, 25, 30, 35, 40, 45, 50 or 100 contiguous nucleotides of saidnucleotide sequence.

In a Fourteenth Aspect: The ligand of any preceding aspect, wherein saiddisease or condition is hyperlipidaemia, hypercholesterolaemia (eg,familial hypercholesterolaemia), heart attack, stroke, coronary heartdisease, atherosclerosis or a cardiovascular disease or condition.

The ligand of any preceding aspect, wherein the disease or condition ishypercholesterolemia, hyperlipidemia, hypercholesterolemia,dyslipidemia, cholestatic liver disease, nephrotic syndrome,hypothyroidism, obesity, atherosclerosis or a cardiovascular disease.

In an example, said disease or condition is hypercholesterolaemia. Theterm “hypercholesterolaemia,” as used herein, refers to a condition inwhich cholesterol levels are elevated above a desired level. In someembodiments, this denotes that serum cholesterol levels are elevated. Insome embodiments, the desired level takes into account various “riskfactors” that are known to one of skill in the art (and are described orreferenced in US20120093818).

The ligand of any preceding aspect, wherein the human is identified asheterozygous for Familial Hypercholesterolemia, statin intolerant,statin uncontrolled, or at risk for developing hypercholesterolemia,dyslipidemia, cholestatic liver disease, nephrotic syndrome,hypothyroidism, obesity, atherosclerosis or a cardiovascular disease.

In a Fifteenth Aspect: The ligand of any preceding aspect, wherein saiddisease or condition is associated with elevated LDL cholesterol.

Cholesterol levels are measured in milligrams (mg) of cholesterol perdeciliter (dL) of blood in the United States and some other countries.Canada and most European countries measure cholesterol in millimoles(mmol) per liter (L) of blood. Below are general guideline ideal rangesand elevated ranges.

Total cholesterol Total cholesterol* (U.S. and some other (Canada andmost countries) of Europe) Below 200 mg/dL Below 5.2 mmol/L Ideal200-239 mg/dL 5.2-6.2 mmol/L Borderline high 240 mg/dL and above Above6.2 mmol/L High LDL cholesterol LDL cholesterol* (U.S. and some other(Canada and most countries) of Europe) 100-129 mg/dL 2.6-3.3 mmol/LIdeal 130-159 mg/dL 3.4-4.1 mmol/L Borderline high 160-189 mg/dL 4.1-4.9mmol/L High 190 mg/dL and above Above 4.9 mmol/L Very high *Canadian andEuropean guidelines differ slightly from U.S. guidelines. Theseconversions are based on U.S. guidelines.

Elevated LDL cholesterol is, therefore, 160 mg/dL or above (4.1 mmol/Lor above).

In a Sixteenth Aspect: The ligand of any preceding aspect, wherein theligand inhibits human PCSK9 binding to human LDL receptor and optionallyhas been or is determined as capable of such inhibition.

In an example, the method comprises (before administering the ligand)determining that the ligand is capable of such inhibition.

Inhibition determination is eg, inhibition in a blood or serum sample,at rtp, at pH7, at 37 degrees centigrade and/or under the physiologicalconditions of a human body.

In a Seventeenth Aspect: The ligand of any preceding aspect, wherein thehuman is resistant or substantially resistant to statin (eg, avorstatinand/or fluvastatin) treatment of said disease or condition.

In an Eighteenth Aspect: The ligand of any preceding aspect, wherein theligand is for treating and/or preventing a PCSK9-mediated disease orcondition in a human

(i) whose genome comprises SEQ ID NO: 29 and wherein the human is ofASW, YRI, GBF, TSL, CLM, LWK, MXL, JPT, PUR, IBS, FIN or CEU ancestry;or(ii) whose genome comprises SEQ ID NO: 30 and wherein the human is ofASW, YRI, GBR, TSI, CLM, CHB, LWK, CHS, JPT, PUR, FIN or CEU ancestry;or(iii) whose genome comprises SEQ ID NO: 32 and wherein the human is ofASW, GBR, TSI, CLM, JPT, PUR, IBS, FIN or CEU ancestry; or(iv) whose genome comprises SEQ ID NO: 33 and wherein the human is ofLWK, ASW, YRI or CLM ancestry; or(v) whose genome comprises SEQ ID NO: 34 and wherein the human is ofLWK, ASW or YRI ancestry; or(vi) whose genome comprises SEQ ID NO: 35 and wherein the human is ofPUR, TSI, FIN or CEU ancestry; or(vii) whose genome comprises SEQ ID NO: 36 and wherein the human is ofLWK, ASW or YRI ancestry; or(viii) whose genome comprises SEQ ID NO: 37 and wherein the human is ofCHS, ASW, JPT, PUR or CHB ancestry.

In a Nineteenth Aspect: The ligand of any preceding aspect, wherein theligand is for treating and/or preventing a PCSK9-mediated disease orcondition in a human

(i) that expresses PCSK9 form f and wherein the human is of ASW, YRI,GBR, TSI, CLM, LWK, MXL, JPT, PUR, IBS, FIN or CEU ancestry; or(ii) that expresses PCSK9 form c and wherein the human is of ASW, YRI,GBR, TSI, CLM, CHB, LWK, CHS, JPT, PUR, FIN or CEU ancestry; or(iii) that expresses PCSK9 form p and wherein the human is of ASW, GBR,TSI, CLM, JPT, PUR, IBS, FIN or CEU ancestry; or(iv) that expresses PCSK9 form m and wherein the human is of LWK, ASW,YRI or CLM ancestry; or(v) that expresses PCSK9 form e and wherein the human is of LWK, ASW orYRI ancestry; or (vi) that expresses PCSK9 form h and wherein the humanis of PUR, TSI, FIN or CEU ancestry; or(vii) that expresses PCSK9 form aj and wherein the human is of LWK, ASWor YRI ancestry; or (viii) that expresses PCSK9 form q and wherein thehuman is of CHS, ASW, JPT, PUR or CHB ancestry.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Twentieth Aspect: A pharmaceutical composition or kit for treatingand/or preventing a PCSK9-mediated condition or disease (eg, as recitedin aspect 14 or 15), the composition or kit comprising a ligand of anypreceding aspect and optionally a statin (eg, cerovastatin,atorvastatin, simvastatin, pitavastin, rosuvastatin, fluvastatin,lovastatin or pravastatin); and optionally in combination with a labelor instructions for use to treat and/or prevent said disease orcondition in a human (eg, covering treatment of a human as recited inaspect 18 or 19); optionally wherein the label or instructions comprisea marketing authorisation number (eg, an FDA or EMA authorisationnumber); optionally wherein the label or instructions comprisedirections to administer alirocumab or evolocumab to said human;optionally wherein the kit comprises an IV or injection device thatcomprises the ligand (and, eg, also a statin).

In a Twenty-first Aspect: A method of producing an anti-human PCSK9antibody binding site, the method comprising obtaining a plurality ofanti-PCSK9 antibody binding sites, screening the antibody binding sitesfor binding to a human PCSK9 selected from the group consisting of formsf, c, r, p, m, e, h, aj and q or a catalytic or C-terminal domain or apeptide thereof that comprises amino acid variation from thecorresponding sequence of SEQ ID NO: 1, 2 or 3 and isolating an antibodybinding site that binds in the screening step, and optionally producinga form f, c, r, p, m, e, h, aj or q PCSK9-binding fragment or derivativeof the isolated antibody.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In an example of this and the next aspect, the plurality of bindingsites comprises or consists of a plurality of 4-chain antibodies orfragments thereof, eg, dAbs, Fabs or scFvs. Suitable methods forproducing pluralities of binding sites for screening include phagedisplay (producing a phage display library of antibody binding sites),ribosome display (producing a ribosome display library of antibodybinding sites), yeast display (producing a yeast display library ofantibody binding sites), or immunisation of a non-human vertebrate (eg,a rodent, eg, a mouse or rat, eg, a Velocimouse™, Kymouse™, Xenomouse™,Aliva Mouse™, HuMab Mouse™, Omnimouse™, Omnirat™ or MeMo Mouse™) with aPCSK9 epitope and isolation of a repertoire of antibody-producing cells(eg, a B-cell, plasma cell or plasmablast repertoire) and/or arepertoire of isolated antibodies.

In an example, the method comprises selecting one or more antibodybinding sites that each specifically binds to a human PCSK9 epitopecomprising amino acid variation from the corresponding sequence of SEQID NO: 1, 2 or 3.

For example, the ligand specifically binds to an epitope comprising anamino acid that is variant compared to the corresponding amino acid ofthe PCSK9 encoded by SEQ ID NO: 1, 2 or 3. In an example, the ligandspecifically binds to an epitope comprising two or more such variantamino acids. In an example, specific binding means binding with anaffinity (Kd) of 1 mM, 100 nM, 10 nM or 1 nM or less, eg, as determinedby SPR.

The term “epitope” is a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

In a Twenty-second Aspect: A method of producing an anti-human PCSK9antibody, the method comprising immunising a non-human vertebrate (eg, amouse or a rat) with a human PCSK9 comprising an amino acid sequenceselected from the group consisting of the amino acid sequences of formsf, c, r, p, m, e, h, aj and q or a catalytic or C-terminal domain or apeptide thereof that comprises amino acid variation from thecorresponding sequence of SEQ ID NO: 1, 2 or 3 and isolating an antibodythat binds a human PCSK9 comprising selected from the group consistingof forms f, c, r, p, m, e, h, aj and q or a catalytic or C-terminaldomain or a peptide thereof that comprises amino acid variation from thecorresponding sequence of SEQ ID NO: 1, 2 or 3, and optionally producinga form f, c, r, p, m, e, h, aj or q PCSK9-binding fragment or derivativeof the isolated antibody.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Twenty-third Aspect: The method of aspect 21 or 22, comprising thestep of obtaining a nucleic acid encoding the antibody, fragment,derivative or binding site and optionally inserting the nucleic acid inan expression vector.

For example, the method comprises isolating a cell (eg, B-cell,plasmablast, plasma cell or memory cell) comprising the nucleic acid,wherein the cell is obtained from a non-human vertebrate that has beenimmunised with the PCSK9 epitope.

In a Twenty-fourth Aspect: A kit for PCSK9 genotyping a human, whereinthe kit comprises a nucleic acid (i) comprising a sequence of 10 or more(eg, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) contiguousnucleotides that specifically hybridises to a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 29-37 or at least thecatalytic domain- or C-terminal domain-encoding sequence thereof, orspecifically hybridises to an antisense sequence or an RNA transcript ofsaid sequence, wherein said sequence of contiguous nucleotideshybridises to at least one nucleotide present in said selected sequencewhich is not present in SEQ ID NO: 28 or hybridises to an antisensesequence or an RNA transcript thereof; and/or (ii) comprising a sequenceof at least 10 or more (eg, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100or more) nucleotides of a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or comprising an antisense sequence orRNA version of said contiguous nucleotides, wherein said sequence ofcontiguous nucleotides comprises at least one nucleotide present in saidselected sequence which is not present in SEQ ID NO: 28.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Twenty-fifth Aspect:

A kit for PCSK9 genotyping or phenotyping a human, wherein the kitcomprises a ligand according to any one of aspects 1 to 19 or anantibody, fragment or derivative produced by the method of any one ofaspects 21 to 23.

In a Twenty-sixth Aspect: Use of an anti-PCSK9 ligand that binds a humanPCSK9 selected from the group consisting of forms f, c, r, p, m, e, h,aj and q in the manufacture of a medicament for treating and/orpreventing a PCSK9-mediated disease or condition in a human whose genomecomprises a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 29-37, optionally for treating and/or preventing aPCSK9-mediated disease or condition in a human as recited in aspect 18or 19.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Twenty-seventh Aspect: Use of an anti-PCSK9 ligand that binds ahuman PCSK9 selected from the group consisting of forms f, c, r, p, m,e, h, aj and q in the manufacture of a medicament for targeting saidPCSK9 in a human to treat and/or prevent a disease or condition mediatedby PCSK9, optionally for targeting PCSK9 in a human as recited in aspect18 or 19.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

The ligand can be any anti-PCSK9 ligand disclosed herein.

In a Twenty-eighth Aspect: The use of aspect 26 or 27, wherein theligand, human, disease or condition is according to any one of aspects 1to 19.

In a Twenty-ninth Aspect: A method of targeting a PCSK9 for treatingand/or preventing a PCSK9-mediated disease or condition in a human, themethod comprising administering an anti-PCSK9 ligand to a humancomprising a nucleotide sequence selected from the group consisting SEQID NOs: 29-37, whereby a PCSK9 encoded by said nucleotide sequence istargeted.

The ligand can be any anti-PCSK9 ligand disclosed herein.

In a Thirtieth Aspect: The method of aspect 29, wherein the methodcomprises targeting a human PCSK9 selected from the group consisting offorms f, c, r, p, m, e, h, aj and q with said ligand to treat and/orprevent said disease or condition in said human.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Thirty-first Aspect: A method of treating and/or preventing adisease or condition mediated by PCSK9 in a human, the method comprisingtargeting a human PCSK9 selected from the group consisting of forms f,c, r, p, m, e, h, aj and q by administering to the human a ligand thatbinds said PCSK9 thereby treating and/or preventing said disease orcondition in the human.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

The ligand can be any anti-PCSK9 ligand disclosed herein.

In a Thirty-second Aspect: The method of aspect 31, wherein the genomeof the human comprises a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Thirty-third Aspect: The method of any one of aspects 29 to 32,wherein the human has been or is genotyped as positive for a nucleotidesequence selected from the group consisting of SEQ ID NOs: 29-37 or thecatalytic- or C-terminal domain-encoding sequence thereof.

In a Thirty-fourth Aspect: The method of any one of aspects 29 to 33,wherein the human has been or is phenotyped as positive for a humanPCSK9 selected from the group consisting of forms f, c, r, p, m, e, h,aj and q.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Thirty-fifth Aspect: The method of any one of aspects 29 to 34,wherein the method comprises genotyping the human as positive for anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37 or the catalytic- or C-terminal domain-encoding sequence thereof.

In a Thirty-sixth Aspect: The method of any one of aspects 29 to 35,wherein the method comprises phenotyping the human as positive for ahuman PCSK9 sequence selected from the group consisting of forms f, c,r, p, m, e, h, aj and q.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Thirty-seventh Aspect: The method of any one of aspects 29 to 36,wherein the human has been or is genotyped as heterozygous for anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37 or the catalytic- or C-terminal domain-encoding sequence thereof;optionally wherein the human has been or is genotyped as comprising thenucleotide sequence of SEQ ID NO: 28 or the catalytic- or C-terminaldomain-encoding sequence thereof and a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 29-37 or the catalytic- orC-terminal domain-encoding sequence thereof.

In a Thirty-eighth Aspect: The method of any one of aspects 29 to 37,wherein the genome of the human has been or is genotyped as homozygousfor a nucleotide sequence selected from the group consisting of SEQ IDNOs: 29-37 or the catalytic- or C-terminal domain-encoding sequencethereof.

In a Thirty-ninth Aspect: The method of any one of aspects 29 to 38,wherein the method comprises genotyping the human for a nucleotidesequence selected from the group consisting of SEQ ID NOs: 29-37 or thecatalytic- or C-terminal domain-encoding sequence thereof beforeadministering the ligand to the human, wherein the ligand is determinedto be capable of binding to a PCSK9 encoded by said selected sequence.

In a Fortieth Aspect: The method of any one of aspects 29 to 39, whereinthe ligand, human, disease or condition is according to any one ofaspects 1 to 19.

In a Forty-first Aspect: A method according to any one of aspects 29 to40 for treating and/or preventing a condition or disease as recited inaspect 14 or 15, the method comprising administering said ligand and astatin (eg, cerovastatin, atorvastatin, simvastatin, pitavastin,rosuvastatin, fluvastatin, lovastatin or pravastatin) to the human.

In a Forty-second Aspect: The method of aspect 41, wherein the ligandand statin are administered separately.

In a Forty-third Aspect: The method of aspect 41, wherein the ligand andstatin are administered simultaneously.

In a Forty-fourth Aspect: The method of any one of aspects 29 to 43,wherein the ligand is administered by subcutaneous injection.

In a Forty-fifth Aspect: A method of PCSK9 genotyping a nucleic acidsample of a human, the method comprising identifying in the sample thepresence of a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 29-37 or the catalytic- or C-terminal domain-encodingsequence thereof.

In a Forty-sixth Aspect: A method of PCSK9 typing a protein sample of ahuman, the method comprising identifying in the sample the presence of ahuman PCSK9 selected from the group consisting of forms f, c, r, p, m,e, h, aj and q.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In an example, the method comprises obtaining a PCSK9 protein samplefrom the human and then carrying out the identifying step.

In a Forty-seventh Aspect: The method of aspect 45 or 46, comprisingobtaining a sample of serum, blood, faeces, hair, tissue, cells, urineor saliva from a human, whereby the nucleic acid or protein sample isobtained and used in the step of identifying said sequence.

In a Forty-eight Aspect: The method of any one of aspects 45 to 47,comprising using a ligand according to any one of aspects 1 to 19 tocarry out said identifying step.

In a Forty-ninth Aspect: A method of treating and/or preventing in ahuman patient a cardiovascular disease or condition, or a disease orcondition that is associated with elevated LDL cholesterol (eg,hypercholesterolaemia), wherein the patient is receiving or haspreviously received statin treatment for said disease or condition, themethod comprising typing the patient using a method of any one ofaspects 45 to 48 and administering a ligand according to one of aspects1 to 19 whereby the human is treated or said disease or condition isprevented; optionally also reducing or stopping statin treatment.

In an example, said reducing or stopping comprises reducing the doseand/or dosing frequency of statin.

In a Fiftieth Aspect: A diagnostic, therapeutic or prophylactic kitcomprising a ligand that is capable of binding to or has been or isdetermined as capable of binding to an amino acid sequence selected fromSEQ ID NOs: 4-27 and instructions for carrying out the method of any oneof aspects 46 to 49 and/or a label or instructions indicating orcovering administration of the ligand to a human as defined in any oneof aspects 1 to 19.

In a Fifty-first Aspect: A diagnostic, therapeutic or prophylactic kitcomprising a nucleic acid probe comprising a nucleotide sequence thatspecifically hybridises to a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or an antisense sequence or RNAtranscript thereof and instructions for carrying out the method ofaspect 45, 47 or 48.

In embodiments of any of the aspects described herein, optionally, thePCSK9 is human PCSK9, eg, a mature, cleaved, autocatalysed or activePCSK9. In an example, the disease is a cardiovascular disease such ashyperlipidaemia.

In examples of the present invention, the ligand specifically binds tohuman PCSK9, eg, one or more of the rare PCSK9 variants disclosed herein(eg, one, two, three, more or all mature forms f, c, r, p, m, e, h, ajand q) and optionally also the a and/or a′ form. For example, the ligandspecifically binds to mature form f and/or c as well as form a.

Determination of such binding can be performed by any antibody bindingtest as known in the art, eg, by surface plasmon resonance. Binding toeach such form is, for example, respectively with a Kd of at least 1 mM,100 nM, 1 nM, 100 pM, 10 pM or 1 pM.

In an example, the ligand binds form a and a PCSK9 selected from thegroup consisting of forms f, c, r, p, m, e, h, aj and q, wherein theligand binding to said selected form is with a Kd (determined by SPR)that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a.In an embodiment, both forms are mature forms. In an embodiment, bothforms are pro-forms.

In an example, the ligand binds form a and form f wherein the ligandbinding to form f is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form c, wherein the ligandbinding to form c is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form r, wherein the ligandbinding to form r is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form p, wherein the ligandbinding to form p is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form m, wherein the ligandbinding to form m is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form e, wherein the ligandbinding to form e is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form h, wherein the ligandbinding to form h is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form aj, wherein the ligandbinding to form aj is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form q, wherein the ligandbinding to form q is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In examples of the present invention, the ligand neutralises humanPCSK9, eg, one or more of the rare PCSK9 variants disclosed herein (eg,one, two, three, more or all mature forms f, c, r, p, m, e, h, aj and q)and optionally also the a and/or a′ form. For example, the ligandneutralises mature form f and/or c as well as form a. Determination ofneutralisation can be performed, for example, by any neutralisationassay method disclosed in US20120093818A1 (Amgen, Inc) orUS20110065902A1 (Regeneron Pharmaceuticals, Inc). Ligands of theinvention that bind or target PCSK9 are useful, for example, fortherapeutic and prophylactic applications disclosed in US20120093818A1and US20110065902A1, these specific disclosures being incorporatedherein by reference in their entirety for use in the present inventionand for possible inclusion in claims herein.

In embodiments where the ligand is used for therapeutic applications, anantigen binding protein can inhibit, interfere with or modulate one ormore biological activities of a PCSK9 (eg, one or more of the rarevariants disclosed herein and optionally also the a and/or a′ form). Inone embodiment, ligand binds specifically to human PCSK9 (eg, one ormore of the rare variants disclosed herein and optionally also the aand/or a′ form) and/or substantially inhibits binding of human PCSK9(eg, said one or more of the rare variants disclosed herein andoptionally also the a and/or a′ form) to LDLR by at least 20%, eg,20%-40%, 40-60%, 60-80%, 80-85%, or more (for example, by measuringbinding in an in vitro competitive binding assay). In an example, theligand is an antibody.

In an embodiment, the ligand has a Kd of less (binding more tightly)than 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ M for binding to one,two or more of the rare variants disclosed herein and optionally alsothe a and/or a′ form. In an example, Kd is determined using SPR.

In an embodiment, the ligand has an IC50 for blocking the binding ofLDLR to one or more of the rare PCSK9 variants disclosed herein (andoptionally also the a and/or a′ form) of less than 1 microM, 1000 nM to100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10pM, 10 pM to 1 pM.

In an embodiment, the ligand has an IC50 for blocking the binding ofLDLR to the a and/or a′ form of PCSK9 that is no more than 1000, 100,90, 80, 70, 60, 50, 40, 30, 20 or 10-fold more (ie, more inhibitory)than the IC50 for blocking the binding of LDLR to one or more of therare PCSK9 variants disclosed herein (eg, one or more PCSK9 proteinscomprising a sequence selected from SEQ ID NOs: 4 to 27). Additionallyor alternatively, for example, the ligand has an IC50 for blocking thebinding of LDLR to (i) the a and/or a′ form of less than 1 microM, 1000nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pMto 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pMto 10 pM, 10 pM to 1 pM, eg, in the range of 1 mM to 1 pM (eg, 1 mM to100 pM; 10 nM to 100 pM; 1 nM to 10 pM; or 100 pM to 1 pM) and (ii) oneor more PCSK9 proteins comprising a sequence selected from SEQ ID NOs: 4to 27 of less than 1 microM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nMto 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pMto 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM, eg, in therange of 1 mM to 1 pM (eg, 1 mM to 100 pM; 10 nM to 100 pM; 1 nM to 10pM; or 100 pM to 1 pM).

In an embodiment, the ligand binds to the a and/or a′ form of PCSK9 witha binding affinity (Kd) that is greater than up to 10%, greater than upto 20%, greater than up to 40%, greater than up to 50%, greater than upto 55%, greater than up to 60%, greater than up to 65%, greater than upto 70%, greater than up to 75%, greater than up to 80%, greater than upto 85%, greater than up to 90%, greater than up to 95% or greater thanup to 100% (ie, is double) relative to binding to a PCSK9 comprising asequence selected from SEQ ID NOs: 4 to 27. Such binding measurementscan be made using a variety of binding assays known in the art, eg,using surface plasmon resonance (SPR), such as by Biacore™ or using theProteOn XPR36™ (Bio-Rad®), or using KinExA® (Sapidyne Instruments, Inc).

In one embodiment, the surface plasmon resonance (SPR) is carried out at25° C. In another embodiment, the SPR is carried out at 37° C.

In one embodiment, the SPR is carried out at physiological pH, such asabout pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (alsoreferred to as HBS-EP)).

In one embodiment, the SPR is carried out at a physiological salt level,eg, 150 mM NaCl.

In one embodiment, the SPR is carried out at a detergent level of nogreater than 0.05% by volume, eg, in the presence of P20 (polysorbate20; eg, Tween-20™) at 0.05% and EDTA at 3 mM.

In one example, the SPR is carried out at 25° C. or 37° C. in a bufferat pH7.6, 150 mM NaCl, 0.05% detergent (eg, P20) and 3 mM EDTA. Thebuffer can contain 10 mM Hepes. In one example, the SPR is carried outat 25° C. or 37° C. in HBS-EP. HBS-EP is available from Teknova Inc(California; catalogue number H8022).

In an example, the affinity of the ligand which is an antibody isdetermined using SPR by 1. Coupling anti-mouse (or other relevantvertebrate) IgG (eg, Biacore BR-1008-38) to a biosensor chip (eg, GLMchip) such as by primary amine coupling; 2. Exposing the anti-mouse IgG(vertebrate antibody) to a test IgG antibody to capture test antibody onthe chip; 3. Passing the test antigen over the chip's capture surface at1024 nM, 256 nM, 64 nM, 16 nM, 4 nM with a 0 nM (i.e. buffer alone); and4. And determining the affinity of binding of test antibody to testantigen using surface plasmon resonance, eg, under an SPR conditiondiscussed above (eg, at 25° C. in physiological buffer). SPR can becarried out using any standard SPR apparatus, such as by Biacore™ orusing the ProteOn XPR36™ (Bio-Rad®).

Regeneration of the capture surface can be carried out with 10 mMglycine at pH1.7. This removes the captured antibody and allows thesurface to be used for another interaction. The binding data can befitted to 1:1 model inherent using standard techniques, eg, using amodel inherent to the ProteOn XPR36™ analysis software.

In an embodiment, assaying or testing of a ligand of the invention iscarried out at or substantially at pH7 (eg, for in vitro tests andassays) and at or substantially at rtp.

One example of an IgG2 heavy chain constant domain of an anti-PCSK9antibody of the present invention has the amino acid sequence as shownin SEQ ID NO: 154, FIG. 3KK of US20120093818A1, which sequence isincorporated herein by reference.

One example of an IgG4 heavy chain constant domain of an anti-PCSK9antibody of the present invention has the amino acid sequence as shownin SEQ ID NO: 155, FIG. 3KK of US20120093818A1, which sequence anddisclosure is incorporated herein by reference in its entirety.

One example of a kappa light chain constant domain of an anti-PCSK9antibody has the amino acid sequence as shown in SEQ ID NO: 157, FIG.3KK of US20120093818A1, which sequence and disclosure is incorporatedherein by reference in its entirety.

One example of a lambda light chain constant domain of an anti-PCSK9antibody has the amino acid sequence as shown in SEQ ID NO: 156, FIG.3KK of US20120093818A1, which sequence and disclosure is incorporatedherein by reference in its entirety.

In examples of the present invention, the ligand binds mature PCSK9, eg,a mature form of one or more of the rare variants disclosed herein andoptionally also the a and/or a′ form.

In examples of the present invention, the ligand binds the catalyticdomain of PCSK9, eg, of a mature form of one or more of the rarevariants disclosed herein and optionally also the a and/or a′ form.

In examples of the present invention, the ligand binds the prodomain ofPCSK9, eg, of a mature form of one or more of the rare variantsdisclosed herein and optionally also the a and/or a′ form.

In some embodiments, the ligand binds to the V domain of PCSK9, eg, of amature form of one or more of the rare variants disclosed herein andoptionally also the a and/or a′ form. In some embodiments, the ligandbinds to the V domain of PCSK9 (eg, of a mature form of one or more ofthe rare variants disclosed herein and optionally also the a and/or a′form) and prevents (or reduces, eg, by at least 10%) PCSK9 from bindingto LDLR. In some embodiments, the ligand binds to the V domain of PCSK9(eg, of a mature form of one or more of the rare variants disclosedherein and optionally also the a and/or a′ form), and while it does notprevent (or reduce) the binding of PCSK9 to LDLR, the ligand prevents orreduces (eg, by at least 10%) the adverse activities mediated throughPCSK9 on LDLR.

In examples of the present invention, the ligand is or comprises a fullyhuman antibody. In an example, the ligand comprises human variableregions or humanised variable regions.

In an example, the ligand of the invention specifically binds to anepitope of a human PCSK9 selected from the group consisting of forms f,c, r, p, m, e, h, aj and q, wherein the epitope comprises at least oneamino acid that is not found in form a. For example, the amino acid isselected from the group consisting of 46L, 53V, 425S, 443T, 474V, 619Pand 670G (numbering as used in SEQ ID NO:1). For example, the amino acidis selected from the group consisting of 425S, 443T, 474V, 619P and 670G(numbering as used in SEQ ID NO:1). For example, the amino acid isselected from the group consisting of 425S and 443T (numbering as usedin SEQ ID NO:1). For example, the amino acid is selected from the groupconsisting of 474V, 619P and 670G (numbering as used in SEQ ID NO:1). Inan example, the PCSK9 form is the mature form. In an example, the PCSK9form is the pro-form. In an example, the ligand also specifically bindsto form a and/or a′. In an embodiment, the ligand specifically binds toan epitope of form fPCSK9, wherein the epitope comprises at least oneamino acid that is not found in form a. In an embodiment, the ligandspecifically binds to an epitope of form c PCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a. In anembodiment, the ligand specifically binds to an epitope of form r PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to an epitopeof form p PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a. In an embodiment, the ligand specificallybinds to an epitope of form m PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a. In an embodiment, theligand specifically binds to an epitope of form e PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to an epitope of form hPCSK9, wherein the epitope comprises at least one amino acid that is notfound in form a. In an embodiment, the ligand specifically binds to anepitope of form aj PCSK9, wherein the epitope comprises at least oneamino acid that is not found in form a. In an embodiment, the ligandspecifically binds to an epitope of form q PCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a.

In an embodiment, ligand binds specifically to the pro-domain of a humanPCSK9 selected from the group consisting of forms f, c, r, p, m, e, h,aj and q. In an example, the ligand also specifically binds to thepro-domain of form a and/or a′. In an embodiment, the ligandspecifically binds to the pro-domain of form fPCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a. In anembodiment, the ligand specifically binds to the pro-domain of form cPCSK9, wherein the epitope comprises at least one amino acid that is notfound in form a. In an embodiment, the ligand specifically binds to thepro-domain of form r PCSK9, wherein the epitope comprises at least oneamino acid that is not found in form a. In an embodiment, the ligandspecifically binds to the pro-domain of form p PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the pro-domain ofform m PCSK9, wherein the epitope comprises at least one amino acid thatis not found in form a. In an embodiment, the ligand specifically bindsto the pro-domain of form e PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a. In an embodiment, theligand specifically binds to the pro-domain of form h PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the pro-domain ofform aj PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a. In an embodiment, the ligand specificallybinds to the pro-domain of form q PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a.

In an embodiment, ligand binds specifically to the catalytic domain of ahuman PCSK9 selected from the group consisting of forms f, c, r, p, m,e, h, aj and q. In an example, the ligand also specifically binds to thecatalytic domain of form a and/or a′. In an embodiment, the ligandspecifically binds to the catalytic domain of form fPCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the catalytic domainof form c PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a. In an embodiment, the ligand specificallybinds to the catalytic domain of form r PCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a. In anembodiment, the ligand specifically binds to the catalytic domain ofform p PCSK9, wherein the epitope comprises at least one amino acid thatis not found in form a. In an embodiment, the ligand specifically bindsto the catalytic domain of form m PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the catalytic domain of form e PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to thecatalytic domain of form h PCSK9, wherein the epitope comprises at leastone amino acid that is not found in form a. In an embodiment, the ligandspecifically binds to the catalytic domain of form aj PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the catalytic domainof form q PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a.

In an embodiment, ligand binds specifically to the C-terminal domain ofa human PCSK9 selected from the group consisting of forms f, c, r, p, m,e, h, aj and q. In an example, the ligand also specifically binds to theC-terminal domain of form a and/or a′. In an embodiment, the ligandspecifically binds to the C-terminal domain of form fPCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the C-terminal domainof form c PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a. In an embodiment, the ligand specificallybinds to the C-terminal domain of form r PCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a. In anembodiment, the ligand specifically binds to the C-terminal domain ofform p PCSK9, wherein the epitope comprises at least one amino acid thatis not found in form a. In an embodiment, the ligand specifically bindsto the C-terminal domain of form m PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the C-terminal domain of form e PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to theC-terminal domain of form h PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a. In an embodiment, theligand specifically binds to the C-terminal domain of form aj PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to theC-terminal domain of form q PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a.

In an embodiment, ligand binds specifically to the substrate-bindinggroove of a human PCSK9 selected from the group consisting of forms f,c, r, p, m, e, h, aj and q (see Cunningham et al., Nat Struct Mol Biol.2007 May; 14(5):413-9. Epub 2007 Apr. 15, “Structural and biophysicalstudies of PCSK9 and its mutants linked to familialhypercholesterolemia”, incorporated herein in its entirety byreference). In an example, the ligand also specifically binds to thesubstrate-binding groove of form a and/or a′. In an embodiment, theligand specifically binds to the Substrate-binding groove of formfPCSK9, wherein the epitope comprises at least one amino acid that isnot found in form a. In an embodiment, the ligand specifically binds tothe Substrate-binding groove of form c PCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a. In anembodiment, the ligand specifically binds to the Substrate-bindinggroove of form r PCSK9, wherein the epitope comprises at least one aminoacid that is not found in form a. In an embodiment, the ligandspecifically binds to the Substrate-binding groove of form p PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to theSubstrate-binding groove of form m PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the Substrate-binding groove of form ePCSK9, wherein the epitope comprises at least one amino acid that is notfound in form a. In an embodiment, the ligand specifically binds to theSubstrate-binding groove of form h PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the Substrate-binding groove of form ajPCSK9, wherein the epitope comprises at least one amino acid that is notfound in form a. In an embodiment, the ligand specifically binds to theSubstrate-binding groove of form q PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a.

Reference is made to US20120093818A1 (Amgen, Inc), the entire disclosureof which is incorporated herein by reference. This patent applicationdiscloses relevant ligands for use in the present invention, as well asexamples and methods of producing and testing ligands that can be usedwith reference to the present invention.

In an example, the ligand is or comprises an antibody disclosed in Table2 of US20120093818A1 (Amgen, Inc) or is a PCSK9-binding derivativethereof.

In an embodiment, the PCSK9-binding ligand of the invention is selectedfrom the antigen binding proteins disclosed in US20120093818A1 (Amgen,Inc), eg, in paragraphs [0009] to [0014] and [0058] to [0063] ofUS20120093818A1; all of these disclosures (including the sequences ofsuch proteins) are incorporated herein by reference as though explicitlyrecited herein and for possible inclusion in one or more claims or foruse in the present invention.

In this paragraph SEQ ID NOs are those as appearing in US20120093818A1(Amgen, Inc) and these sequences are incorporated herein by reference asthough explicitly recited herein and for possible inclusion in one ormore claims or for use in the present invention. In some aspects, theligand of the invention comprises an isolated antigen binding proteinthat binds PCSK9 comprising: A) one or more heavy chain complementarydetermining regions (CDRHs) selected from the group consisting of: (i) aCDRH1 from a CDRH1 in a sequence selected from the group consisting ofSEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49,57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; (ii)a CDRH2 from a CDRH2 in a sequence selected from the group consisting ofSEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49,57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60;(iii) a CDRH3 from a CDRH3 in a sequence selected from the groupconsisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54,55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81,and 60; and (iv) a CDRH of (i), (ii), and (iii) that contains one ormore amino acid substitutions, deletions or insertions of no more than 4amino acids; B) one or more light chain complementary determiningregions (CDRLs) selected from the group consisting of: (i) a CDRL1 froma CDRL1 in a sequence selected from the group consisting of SEQ ID NO:5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30,31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; (ii) a CDRL2 from aCDRL2 in a sequence selected from the group consisting of SEQ ID NO: 5,7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30,31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; (iii) a CDRL3 from aCDRL3 in a sequence selected from the group consisting of SEQ ID NO: 5,7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30,31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; and (iv) a CDRL of(i), (ii) and (iii) that contains one or more amino acid substitutions,deletions or insertions of no more than 4 amino acids; or C) one or moreheavy chain CDRHs of A) and one or more light chain CDRLs of B). In someembodiments, the isolated antigen binding protein comprises at least oneCDRH of A) and at least one CDRL of B). In some embodiments, theisolated antigen binding protein comprises at least two CDRH of A) andat least two CDRL of B). In some embodiments, the isolated antigenbinding protein comprises said CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 andCDRL3. In some embodiments, the CDRH of A) is selected from at least oneof the group consisting of: (i) a CDRH1 amino acid sequence selectedfrom the CDRH1 in a sequence selected from the group consisting of SEQID NO: 67, 79, 89, and 49; (ii) a CDRH2 amino acid sequence selectedfrom the CDRH2 in a sequence selected from the group consisting of SEQID NO: 67, 79, 89, and 49; (iii) a CDRH3 amino acid sequence selectedfrom the CDRH3 in a sequence selected from the group consisting of SEQID NO: 67, 79, 89, and 49; and (iv) a CDRH of (i), (ii) and (iii) thatcontains one or more amino acid substitutions, deletions or insertionsof no more than 2 amino acids. In addition, the CDRL of B) is selectedfrom at least one of the group consisting of: (i) a CDRL1 amino acidsequence selected from the CDRL1 in a sequence selected from the groupconsisting of SEQ ID NO: 12, 35, 32, and 23; (ii) a CDRL2 amino acidsequence selected from the CDRL2 in a sequence selected from the groupconsisting of SEQ ID NO: 12, 35, 32, and 23; (iii) a CDRL3 amino acidsequence selected from the CDRL3 in a sequence selected from the groupconsisting of SEQ ID NO: 12, 35, 32, and 23; and (iv) a CDRL of (i),(ii) and (iii) that contains one or more amino acid substitutions,deletions or insertions of no more than 2 amino acids; or C) one or moreheavy chain CDRHs of A) and one or more light chain CDRLs of B. In someembodiments, the CDRH of A) is selected from at least one of the groupconsisting of: (i) a CDRH1 amino acid sequence of the CDRH1 amino acidsequence in SEQ ID NO: 67; (ii) a CDRH2 amino acid sequence of the CDRH2amino acid sequence in SEQ ID NO: 67; (iii) a CDRH3 amino acid sequenceof the CDRH3 amino acid sequence in SEQ ID NO: 67; and (iv) a CDRH of(i), (ii) and (iii) that contains one or more amino acid substitutions,deletions or insertions of no more than 2 amino acids; said CDRL of B)is selected from at least one of the group consisting of: (i) a CDRL1amino acid sequence of the CDRL1 amino acid sequence in SEQ ID NO: 12;(ii) a CDRL2 amino acid sequence of the CDRL2 amino acid sequence in SEQID NO: 12; (iii) a CDRL3 amino acid sequence of the CDRL3 amino acidsequence in SEQ ID NO: 12; and (iv) a CDRL of (i), (ii) and (iii) thatcontains one or more amino acid substitutions, deletions or insertionsof no more than 2 amino acids; or C) one or more heavy chain CDRHs of A)and one or more light chain CDRLs of B). In some embodiments, theantigen binding protein comprises A) a CDRH1 of the CDRH1 sequence inSEQ ID NO: 67, a CDRH2 of the CDRH2 sequence in SEQ ID NO: 67, and aCDRH3 of the CDRH3 sequence in SEQ ID NO: 67, and B) a CDRL1 of theCDRL1 sequence in SEQ ID NO: 12, a CDRL2 of the CDRL2 sequence in SEQ IDNO: 12, and a CDRL3 of the CDRL3 sequence in SEQ ID NO: 12. In someembodiments, the antigen binding protein comprises a heavy chainvariable region (VH) having at least 80% sequence identity with an aminoacid sequence selected from the group consisting of SEQ ID NO: 74, 85,71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62,89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or a light chainvariable region (VL) having at least 80% sequence identity with an aminoacid sequence selected from the group consisting of SEQ ID NO: 5, 7, 9,10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32,33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In some embodiments, the VHhas at least 90% sequence identity with an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52,51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77,78, 83, 69, 81, and 60, and/or the VL has at least 90% sequence identitywith an amino acid sequence selected from the group consisting of SEQ IDNO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28,30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In someembodiments, the VH is selected from the group consisting of SEQ ID NO:74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91,64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or the VL isselected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36,37, 38, 39, 40, 42, 44, and 46.

In an example of any aspect of the invention, the PCSK9-targeting orbinding ligand comprises or consists of AMG145 or 31H4, 16F12, 11F1, 8A3or 21B12 disclosed in US20120093818A1 (Amgen, Inc) or an antibodycomprising the variable domains of AMG145, 31H4, 16F12, 11F1, 8A3 or21B12, the disclosures of which (including sequences) are incorporatedherein by reference as though explicitly recited herein and for possibleinclusion in one or more claims or for use in the present invention.Preferably, the PCSK9-targeting or binding ligand comprises or consistsof AMG145.

In an example, the AMG145 or other ligand of the invention isglycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos orHek293 cell). In an example, the ligand of the invention is produced inCHO.

Reference is made to US20110065902A1 (Regeneron Pharmaceuticals, Inc),the entire disclosure of which is incorporated herein by reference. Thispatent application discloses relevant ligands for use in the presentinvention, as well as examples and methods of producing and testingligands and determining medical efficacy that can be used with referenceto the present invention.

Reference is made to the following PCT applications, the entiredisclosures of which are incorporated herein by reference. Thesedisclose relevant ligands for use in the present invention, as well asexamples and methods of producing and testing ligands and determiningmedical efficacy that can be used with reference to the presentinvention.

-   WO2008057457-   WO2008057458-   WO2008057459-   WO2008063382-   WO2008133647-   WO2009100297-   WO2009100318-   WO2011037791-   WO2011053759-   WO2011053783-   WO2008125623-   WO2011072263-   WO2009055783-   WO2010029513-   WO2011111007-   WO2010077854 Antibody ligands to PCSK9 are described in, for    example, WO 2008/057457, WO 2008/057458, WO 2008/057459, WO    2008/063382, WO 2008/125623, and US 2008/0008697.

In an example, the ligand is or comprises an antibody disclosed in theExamples of US20110065902A1 (eg, 316P or 300N) or is a PCSK9-bindingderivative thereof. All of these disclosures (including the sequences ofsuch proteins and corresponding nucleotide sequences) are incorporatedherein by reference as though explicitly recited herein and for possibleinclusion in one or more claims or for use in the present invention. Inan embodiment, the ligand is or comprises the variable domains ofantibody 316P or 300N disclosed in US20110065902A1 or is (or comprises)such antibody or a PCSK9-binding derivative thereof. The foregoingreference is incorporated by reference herein in its entirety.

In an embodiment, the ligand is or comprises the variable domains ofantibody alirocumab or SAR236553/REGN727 (Sanofi Aventis/Regeneron) oris (or comprises) such antibody or a PCSK9-binding derivative thereof.In an example, the antibody is glycosylated, eg, has human glycosylation(eg, produced by a CHO, Cos or Hek293 cell). Preferably, the ligand isalirocumab or SAR236553/REGN727.

In an embodiment, the ligand is or comprises the variable domains ofantibody evolocumab or is (or comprises) such antibody or aPCSK9-binding derivative thereof. In an example, the antibody isglycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos orHek293 cell). Preferably, the ligand is evolocumab.

In an embodiment, the ligand is selected from evolocumab, 1D05-IgG2(Merck & Co.), ALN-PCS02 (Alnylam), RN316 (Pfizer-Rinat) and alirocumab.

In an embodiment, the ligand is selected from the following (sequencesand definitions as per US2011/0065902, incorporated herein by referencein its entirety):—

1. An antibody or antigen-binding fragment thereof which specificallybinds hPCSK9, wherein the antibody or antigen-binding fragment comprisesthe heavy and light chain CDRs of a HCVR/LCVR amino acid sequence pairhaving SEQ ID NOs: 218/226.2. The antibody or antigen-binding fragment of concept 1 comprisingheavy and light chain CDR amino acid sequences having SEQ ID NOs: 220,222, 224, 228, 230 and 232.3. The antibody or antigen-binding fragment of concept 2 comprising anHCVR having the amino acid sequence of SEQ ID NO: 218 and an LCVR havingthe amino acid sequence of SEQ ID NO: 226.4. An antibody or antigen-binding fragment thereof which binds to thesame epitope on hPCSK9 as an antibody comprising heavy and light chainCDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and232.5. An antibody or antigen-binding fragment thereof which competes forbinding to hPCSK9 with an antibody comprising heavy and light chain CDRamino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232.

In an embodiment, the ligand is selected from the following (sequencesand definitions as per US2012/0093818, incorporated herein by referenceherein in its entirety):—

1. An isolated neutralizing antigen binding protein that binds to aPCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1,wherein the neutralizing antigen binding protein decreases the LDLRlowering effect of PCSK9 on LDLR, wherein the antigen binding proteincomprises a light chain comprising an amino acid sequence of SEQ ID NO:46, and wherein the antigen binding protein comprises a heavy chaincomprising an amino acid sequence of SEQ ID NO: 60.2. The isolated neutralizing antigen binding protein of concept 2,wherein the antigen binding protein is a LDLR non-competitiveneutralizing antigen binding protein.3. The isolated neutralizing antigen binding protein of concept 2,wherein the antigen binding protein is a LDLR competitive neutralizingantigen binding protein.4. An antigen binding protein that selectively binds to PCSK9, whereinsaid antigen binding protein binds to PCSK9 with a Kd that is less than100 pM.5. An antigen binding protein that binds to a PCSK9 protein of SEQ IDNO: 303 in a first manner, wherein the antigen binding protein binds toa variant of PCSK9 in a second manner, wherein said PCSK9 variant has atleast one point mutation at a position selected from the groupconsisting of: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390,413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554 ofSEQ ID NO: 303, wherein the first manner comprises a first EC50, a firstBmax, or a first EC50 and a first Bmax, wherein the second mannercomprises a second EC50, a second Bmax, or a second EC50 and a secondBmax, and wherein a value for the first manner is different from a valuefor the second manner, and wherein the antigen binding protein comprisesa light chain comprising an amino acid sequence of SEQ ID NO: 46, andwherein the antigen binding protein comprises a heavy chain comprisingan amino acid sequence of SEQ ID NO: 60.6. The antigen binding protein of concept 6, wherein the first mannercomprises a first Bmax, wherein the second manner comprises a secondBmax that is different from the first Bmax, and wherein said PCSK9variant has at least one point mutation selected from the groupconsisting of: D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R,R519E, H521R, and Q554R.7. The antigen binding protein of concept 6, wherein the antigen bindingprotein binds to PCSK9 at a location that overlaps with a location thatLDLR binds to PCSK9.8. A method of making an antigen binding protein that binds to a PCSK9protein comprising the amino acid sequence of SEQ ID NO: 1, wherein theantigen binding protein decreases the LDLR lowering effect of PCSK9 onLDLR, said method comprising: providing a host cell comprising a nucleicacid sequence that encodes the antigen binding protein; and maintainingthe host cell under conditions in which the antigen binding protein isexpressed, wherein the antigen binding protein comprises a light chaincomprising an amino acid sequence of SEQ ID NO: 46, and wherein theantigen binding protein comprises a heavy chain comprising an amino acidsequence of SEQ ID NO: 60.9. A method for treating or preventing a condition associated withelevated serum cholesterol levels in a subject, said method comprisingadministering to a subject in need thereof an effective amount of anisolated neutralizing antigen binding protein simultaneously orsequentially with an agent that elevates the availability of LDLRprotein, wherein the isolated antigen binding protein binds to a PCSK9protein comprising the amino acid sequence of SEQ ID NO: 1, wherein theneutralizing antigen binding protein decreases the LDLR lowering effectof PCSK9 on LDLR, wherein the antigen binding protein comprises a lightchain comprising an amino acid sequence of SEQ ID NO: 46, and whereinthe antigen binding protein comprises a heavy chain comprising an aminoacid sequence of SEQ ID NO: 60.10. The method of concept 10, wherein the agent that elevates theavailability of LDLR protein comprises a statin.11. An antigen binding protein that binds to PCSK9, wherein when theantigen binding protein is bound to PCSK9, the antibody is positioned 8angstroms or less from at least one of the following residues of PCSK9:S153, S188, 1189, Q190, S191, D192, R194, E197, G198, R199, V200, D224,R237, D238, K243, S373, D374, S376, T377, F379, 1154, T187, H193, E195,1196, M201, V202, C223, T228, S235, G236, A239, G244, M247, 1369, S372,C375, or C378, wherein the antigen binding protein comprises a lightchain comprising an amino acid sequence of SEQ ID NO: 46, and whereinthe antigen binding protein comprises a heavy chain comprising an aminoacid sequence of SEQ ID NO: 60.

The ligand can be used for the treatment, therapy, prophylaxis and/ordiagnosis of one or more diseases or conditions or susceptibilitythereto, wherein such diseases or conditions comprise those disclosed inUS20120093818A1 (Amgen, Inc) and US20110065902A1 (RegeneronPharmaceuticals, Inc), eg, a disease or condition disclosed inparagraphs [0375] to [0383] of US20120093818A1, which disclosure isincorporated herein by reference in its entirety for inclusion in onemore claims herein.

The ligand can be administered to a human characterised as described inUS20120093818A1 (Amgen, Inc) or US20110065902A1; each of which isincorporated by reference herein in its entirety.

The ligand can be administered in a form or combination disclosed inUS20120093818A1 (Amgen, Inc) or US20110065902A1, which disclosure isincorporated herein by reference. For example, the ligand with a drug,excipient, diluent or carrier as described in US20120093818A1 (Amgen,Inc) or US20110065902A1 (eg, as disclose in paragraphs [0384] to [0412]of US20120093818A1), which disclosure is incorporated herein byreference, and the present invention also relates to the correspondingpharmaceutical compositions comprising the combination of a ligand ofthe invention and such a further agent. Each of the foregoing referencesis incorporated by reference herein in its entirety.

The ligand can be used in a method of diagnosis as set out inUS20120093818A1 (Amgen, Inc) or US20110065902A1, eg, in paragraphs[0413] to [0415] of US20120093818A1 which disclosure is incorporatedherein by reference. Each of the foregoing references is incorporated byreference herein in its entirety.

Diagnostic Applications

In some embodiments, the ligand of the invention is a diagnostic tool.The ligand can be used to assay the amount of PCSK9 present in a sampleand/or subject. As will be appreciated by one of skill in the art, suchligands need not be neutralizing ligands. In some embodiments, thediagnostic ligand is not a neutralizing ligand. In some embodiments, thediagnostic ligand binds to a different epitope than a neutralizingligand binds to. In some embodiments, the two ligands do not competewith one another.

In some embodiments, the ligands of the invention are used or providedin an assay kit and/or method for the detection of PCSK9 in mammaliantissues or cells in order to screen/diagnose for a disease or disorderassociated with changes in levels of PCSK9. The kit comprises a ligandthat binds PCSK9 and means for indicating the binding of the ligand withPCSK9, if present, and optionally PCSK9 protein levels. Various meansfor indicating the presence of a ligand can be used. For example,fluorophores, other molecular probes, or enzymes can be linked to theligand and the presence of the ligand can be observed in a variety ofways. The method for screening for such disorders can involve the use ofthe kit, or simply the use of one of the disclosed ligands and thedetermination of whether the ligand binds to PCSK9 in a sample. As willbe appreciated by one of skill in the art, high or elevated levels ofPCSK9 will result in larger amounts of the ligand binding to PCSK9 inthe sample. Thus, degree of ligand binding can be used to determine howmuch PCSK9 is in a sample. Subjects or samples with an amount of PCSK9that is greater than a predetermined amount (e.g., an amount or rangethat a person without a PCSK9 related disorder would have) can becharacterized as having a PCSK9 mediated disorder. In some embodiments,the invention provides a method wherein the ligand is administered to asubject taking a statin, in order to determine if the statin hasincreased the amount of PCSK9 in the subject.

In some embodiments, the ligand is a non-neutralizing ligand and is usedto determine the amount of PCSK9 in a subject receiving an ABP and/orstatin treatment.

In some embodiments, the ligand of the invention can specifically bindhuman PCSK9 (eg, one, two or more rare variant forms disclosed herein)and is characterized by at least one of: (i) capable of reducing serumtotal cholesterol at least about 25-35% and sustaining the reductionover at least a 24 day period relative to a predose level; (ii) capableof reducing serum LDL cholesterol at least about 65-80% and sustainingthe reduction over at least a 24 day period relative to a predose level;(iii) capable of reducing serum LDL cholesterol at least about 40-70%and sustaining the reduction over at least a 60 or 90 day periodrelative to a predose level; (iv) capable of reducing serum triglycerideat least about 25-40% relative to predose level; (v) does not reduceserum HDL cholesterol or reduces serum HDL cholesterol no more than 5%relative to predose level. In some embodiments, an isolated nucleic acidmolecule is provided and it encodes the ligand. In some embodiments anexpression vector is provided and comprises the nucleic acid molecule.In some embodiments, a pharmaceutical composition is provided and it cancomprise the ligand and a pharmaceutically acceptable carrier. In someembodiments, a method is provided for treating a disease or conditionwhich is ameliorated, improved, inhibited or prevented with a PCSK9antagonist ligand of the invention. The method can compriseadministering a therapeutic amount of the pharmaceutical composition orligand to a subject in need thereof. In some embodiments, the subject isa human subject suffering from hypercholesterolemia, hyperlipidemia,indicated for LDL apheresis, identified as heterozygous for FamilialHypercholesterolemia, statin intolerant. statin uncontrolled, at riskfor developing hypercholesterolemia, dyslipidemia, cholestatic liverdisease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosisand cardiovascular diseases. In some embodiments, a method of providinga treatment or therapy is provided to a subject. In some embodiments,the method comprises reducing serum cholesterol at least about 40-70%over at least 60 to 90 days. In some embodiments, a method of receivingtreatment or therapy is provided, the method can comprise receiving aligand thereof at a frequency of once every 60 to 90 days.

In one aspect, the invention provides a ligand of the invention which isor comprises an human antibody or antigen-binding fragment of a humanantibody that specifically binds and inhibits human proproteinconvertase subtilisin/kexin type 9 (hPCSK9, eg, one, two or more rarevariant forms disclosed herein and optionally form a and/or form a′),characterized by the ability to reduce serum LDL cholesterol in a humanby 40-80% over a 24, 60 or 90 day period relative to predose levels,with little or no reduction in serum HDL cholesterol and/or with littleor no measurable effect on liver function, as determined by ALT and ASTmeasurements.

In one embodiment, the ligand of the invention comprises an antibody orantigen-binding fragment of an antibody that specifically binds hPCSK9and is characterized by at least one of:

(i) capable of reducing serum total cholesterol at least about 25-35%and sustaining the reduction over at least a 24 day period relative to apredose level, preferably the reduction in serum total cholesterol is atleast about 30-40%;(ii) capable of reducing serum LDL cholesterol at least about 65-80% andsustaining the reduction over at least a 24 day period relative to apredose level;(iii) capable of reducing serum triglyceride at least about 25-40%relative to predose level;(iv) does not reduce serum HDL cholesterol or reduces serum HDLcholesterol no more than 5% relative to predose level.See US2011/0065902 for definitions of these terms and optional features,the disclosure of which are incorporated herein by reference in itsentirety.

In one embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody that specifically binds hPCSK9and is characterized by at least one of:

(i) capable of reducing serum LDL cholesterol at least about 40-70% andsustaining the reduction over at least a 60 or 90 day period relative toa predose level;(ii) capable of reducing serum triglyceride at least about 25-40%relative to predose level;(iii) does not reduce serum HDL cholesterol or reduces serum HDLcholesterol no more than 5% relative to predose level.

In one embodiment, the antibody or antigen-binding fragment ischaracterized as exhibiting an enhanced binding affinity (KD) for hPCSK9at pH 5.5 relative to the KD at pH 7.4, as measured by plasmon surfaceresonance. In a specific embodiment, the antibody or fragment thereofexhibits at least a 20-fold, at least a 40-fold or at least a 50-foldenhanced affinity for PCSK9 at an acidic pH relative to a neutral pH, asmeasured by surface plasmon resonance.

In one embodiment, the antibody or antigen-binding fragment ischaracterized as not exhibiting an enhanced binding affinity for PCSK9at an acidic pH relative to a neutral pH, as measured by surface plasmonresonance. In a specific embodiment, the antibody or fragment thereofexhibits a decreased binding affinity at an acidic pH.

In another embodiment, the antibody or antigen-binding fragment bindshuman, human GOF mutation D374Y, cynomolgus monkey, rhesus monkey,mouse, rat and hamster PCSK9.

In one embodiment, the antibody or antigen-binding fragment binds humanand monkey PCSK9, but does not bind mouse, rat or hamster PCSK9.

In one embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody comprising one or more of aheavy chain variable region (HCVR), light chain variable region (LCVR),HCDR1, HCDR2, HCDR3 disclosed in any of paragraphs [023]-[037] ofUS2011/0065902, the disclosure of which is incorporated herein byreference in its entirety.

In a related embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody which specifically binds hPCSK9,wherein the antibody or fragment comprises heavy and light chain CDRdomains contained within heavy and light chain sequence pairs selectedfrom the group consisting of SEQ ID NO (using the sequence numbering inUS2011/0065902): 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68,70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140,142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194/202,210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264,266/274, 282/284, 286/288, 290/298, 306/308, 310/312, 314/322, 330/332,334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384, 386/394,402/404, 406/408, 410/418, 426/428, 430/432, 434/442, 450/452, 454/456,458/466, 474/476, 478/480, 482/490, 498/500, 502/504, 506/514, 522/524,526/528, 530/538, 546/548, 550/552, 554/562, 570/572, 574/576, 578/586,594/596, 598/600, 602/610, 618/620, 622/624, 626/634, 642/644, 646/648,650/658, 666/668, 670/672, 674/682, 690/692, 694/696, 698/706, 714/716,718/720, 722/730, 738/740 and 742/744. In one embodiment, the CDRsequences are contained within HCVR and LCVR selected from the aminoacid sequence pairs of SEQ ID NO: 50/58, 66/68, 70/72, 74/82, 90/92,94/96, 122/130, 138/140, 142/144, 218/226, 234/236, 238/240, 242/250,258/260, 262/264, 314/322, 330/332 and 334/336. In more specificembodiments, the CDR sequences are comprised within HCVR/LCVR sequencesselected from SEQ ID NO: 90/92 or 218/226. Each of the foregoingreferences is incorporated by reference herein in its entirety.

In an example, the invention features a pharmaceutical compositioncomprising a ligand of the invention, wherein the ligand comprises orconsists of a recombinant human antibody or fragment thereof whichspecifically binds hPCSK9 and a pharmaceutically acceptable carrier. Inone embodiment, the invention features a composition which is acombination of a ligand of the invention (eg, an antibody orantigen-binding fragment of an antibody), and a second therapeuticagent. The second therapeutic agent may be any agent that isadvantageously combined with the ligand of the invention, for example,an agent capable of inducing a cellular depletion of cholesterolsynthesis by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A(CoA) reductase, such as, for example, cerovastatin, atorvastatin,simvastatin, pitavastin, rosuvastatin, fluvastatin, lovastatin,pravastatin, etc; capable of inhibiting cholesterol uptake and or bileacid re-absorption; capable of increasing lipoprotein catabolism (suchas niacin); and/or activators of the LXR transcription factor that playsa role in cholesterol elimination such as 22-hydroxycholesterol.

In an example, the invention provides a method for inhibiting hPCSK9activity using the anti-PCSK9 ligand of the invention (eg, an antibodyor antigen-binding portion of the antibody of the invention), whereinthe therapeutic methods comprise administering a therapeuticallyeffective amount of a pharmaceutical composition comprising an antibodyor antigen-binding fragment of an antibody of the invention. Thedisorder treated is any disease or condition which is improved,ameliorated, inhibited or prevented by removal, inhibition or reductionof PCSK9 activity. Specific populations treatable by the therapeuticmethods of the invention include subjects indicated for LDL apheresis,subjects with PCSK9-activating mutations (gain of function mutations,“GOF”), subjects with heterozygous Familial Hypercholesterolemia (heFH);subjects with primary hypercholesterolemia who are statin intolerant orstatin uncontrolled; and subjects at risk for developinghypercholesterolemia who may be preventably treated. Other indicationsinclude dyslipidemia associated with secondary causes such as Type 2diabetes mellitus, cholestatic liver diseases (primary biliarycirrhosis), nephrotic syndrome, hypothyroidism, obesity; and theprevention and treatment of atherosclerosis and cardiovascular diseases.

In specific embodiments of the method of the invention, the ligand ofthe invention (eg, anti-hPCSK9 antibody or antibody fragment of theinvention) is useful to reduce elevated total cholesterol, non-HDLcholesterol, LDL cholesterol, and/or apolipoprotein B (apolipoproteinB100).

The ligand (eg, antibody or antigen-binding fragment) of the inventionmay be used alone or in combination with a second agent, for example, anHMG-CoA reductase inhibitor and/or another lipid lowering drug.

The term “isolated” with reference to a ligand, antibody or protein, forexample in any aspect, configuration, example or embodiment, means thata subject ligand, antibody, protein etc (1) is free of at least someother proteins with which it would normally be found, (2) is essentiallyfree of other proteins from the same source, e.g., from the samespecies, (3) is expressed by a cell from a different species, (4) hasbeen separated from at least about 50 percent of polynucleotides,lipids, carbohydrates, or other materials with which it is associated innature, (5) is operably associated (by covalent or noncovalentinteraction) with a polypeptide with which it is not associated innature, or (6) does not occur in nature. Typically, an “isolated”ligand, antibody, protein etc constitutes at least about 5%, at leastabout 10%, at least about 25%, or at least about 50% of a given sample.Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or anycombination thereof can encode such an isolated ligand, antibody proteinetc. Preferably, the isolated ligand, antibody protein etc issubstantially free from proteins or polypeptides or other contaminantsthat are found in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic, research or other use.

For example, an “isolated” antibody is one that has been identified,separated and/or recovered from a component of its productionenvironment (eg, naturally or recombinantly). Preferably, the isolatedpolypeptide is free of association with all other components from itsproduction environment, eg, so that the antibody has been isolated to anFDA-approvable or approved standard. Contaminant components of itsproduction environment, such as that resulting from recombinanttransfected cells, are materials that would typically interfere withresearch, diagnostic or therapeutic uses for the antibody, and mayinclude enzymes, hormones, and other proteinaceous or non-proteinaceoussolutes. In preferred embodiments, the polypeptide will be purified: (1)to greater than 95% by weight of antibody as determined by, for example,the Lowry method, and in some embodiments, to greater than 99% byweight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under non-reducing orreducing conditions using Coomassie blue or, preferably, silver stain.Isolated antibody includes the antibody in situ within recombinant cellssince at least one component of the antibody's natural environment willnot be present. Ordinarily, however, an isolated polypeptide or antibodywill be prepared by at least one purification step.

Immunoconjugates

The invention encompasses the ligand (eg, antibody) conjugated to atherapeutic moiety (“immunoconjugate”), such as a cytotoxin, achemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxinagents include any agent that is detrimental to cells. Examples ofsuitable cytotoxin agents and chemotherapeutic agents for formingimmunoconjugates are known in the art, see for example, WO 05/103081,which is incorporated by reference herein in its entirety.

Bispecifics

The antibodies of the present invention may be monospecific, bispecific,or multispecific. Multispecific mAbs may be specific for differentepitopes of one target polypeptide or may contain antigen-bindingdomains specific for more than one target polypeptide. See, e.g., Tuttet al. (1991) J. Immunol. 147:60-69. The human anti-PCSK9 (eg,anti-PCSK9) mAbs can be linked to or co-expressed with anotherfunctional molecule, e.g., another peptide or protein. For example, anantibody or fragment thereof can be functionally linked (e.g., bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other molecular entities, such as another antibody orantibody fragment, to produce a bispecific or a multispecific antibodywith a second binding specificity.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)CH3 domain and a second Ig CH3 domain, wherein the first and second IgCH3 domains differ from one another by at least one amino acid, andwherein at least one amino acid difference reduces binding of thebispecific antibody to Protein A as compared to a bi-specific antibodylacking the amino acid difference. In one embodiment, the first Ig CH3domain binds Protein A and the second Ig CH3 domain contains a mutationthat reduces or abolishes Protein A binding such as an H95R modification(by IMGT exon numbering; H435R by EU numbering). The second CH3 mayfurther comprise a Y96F modification (by IMGT; Y436F by EU). Furthermodifications that may be found within the second CH3 include: D16E,L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N,V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, andV82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT;Q355R, N3845, K392N, V397M, R409K, E419Q, and V422I by EU) in the caseof IgG4 antibodies. Variations on the bi-specific antibody formatdescribed above are contemplated within the scope of the presentinvention.

Treatment Population

The invention provides therapeutic methods for treating a human patientin need of a composition or ligand of the invention. While modificationsin lifestyle and conventional drug treatment are often successful inreducing cholesterol levels, not all patients are able to achieve therecommended target cholesterol levels with such approaches. Variousconditions, such as familial hypercholesterolemia (FH), appear to beresistant to lowering of LDL-C levels in spite of aggressive use ofconventional therapy. Homozygous and heterozygous familialhypercholesterolemia (hoFH, heFH) is a condition associated withpremature atherosclerotic vascular disease. However, patients diagnosedwith hoFH are largely unresponsive to conventional drug therapy and havelimited treatment options. Specifically, treatment with statins, whichreduce LDL-C by inhibiting cholesterol synthesis and upregulating thehepatic LDL receptor, may have little effect in patients whose LDLreceptors are non-existent or defective. A mean LDL-C reduction of onlyless than about 20% has been recently reported in patients withgenotype-confirmed hoFH treated with the maximal dose of statins. Theaddition of ezetimibe 10 mg/day to this regimen resulted in a totalreduction of LDL-C levels of 27%, which is still far from optimal.Likewise, many patients are statin non-responsive, poorly controlledwith statin therapy, or cannot tolerate statin therapy; in general,these patients are unable to achieve cholesterol control withalternative treatments. There is a large unmet medical need for newtreatments that can address the short-comings of current treatmentoptions.

Specific populations treatable by the therapeutic methods of theinvention include patients indicated for LDL apheresis, subjects withPCSK9-activating (GOF) mutations, heterozygous FamilialHypercholesterolemia (heFH); subjects with primary hypercholesterolemiawho are statin intolerant or statin uncontrolled; and subjects at riskfor developing hypercholesterolemia who may be preventably treated.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising theanti-PCSK9 ligands, antibodies or antigen-binding fragments thereof ofthe present invention. The administration of therapeutic compositions inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTINT™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. See also Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose may vary depending upon the age and the size of a subject to beadministered, target disease, conditions, route of administration, andthe like. When the ligand, eg, antibody, of the present invention isused for treating various conditions and diseases associated with PCSK9,including hypercholesterolemia, disorders associated with LDL andapolipoprotein B, and lipid metabolism disorders, and the like, in anadult patient, it is advantageous to intravenously administer the ligandor antibody of the present invention normally at a single dose of about0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight.Depending on the severity of the condition, the frequency and theduration of the treatment can be adjusted.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, thus the compositioninvention provides the ligand by e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al.(1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include,but are not limited to, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes. Thecomposition may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533; Treat etal. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365;Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138,1984).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule. Apharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT™,OPTIPEN PRO™, OPTIPEN STARLETT™, and OPTICLIKT™ (sanofi-aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

The invention provides therapeutic methods in which the ligand, eg,antibody or antibody fragment, of the invention is useful to treathypercholesterolemia associated with a variety of conditions involvinghPCSK9. The anti-PCSK9 ligands, eg, antibodies or antibody fragments, ofthe invention are particularly useful for the treatment ofhypercholesterolemia and the like. Combination therapies may include theanti-PCSK9 ligand of the invention with, for example, one or more of anyagent that (1) induces a cellular depletion of cholesterol synthesis byinhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase,such as cerivastatin, atorvastatin, simvastatin, pitavastatin,rosuvastatin, fluvastatin, lovastatin, pravastatin; (2) inhibitscholesterol uptake and or bile acid re-absorption; (3) increaselipoprotein catabolism (such as niacin); and activators of the LXRtranscription factor that plays a role in cholesterol elimination suchas 22-hydroxycholesterol or fixed combinations such as ezetimibe plussimvastatin; a statin with a bile resin (e.g., cholestyramine,colestipol, colesevelam), a fixed combination of niacin plus a statin(e.g., niacin with lovastatin); or with other lipid lowering agents suchas omega-3-fatty acid ethyl esters (for example, omacor).

Ligands of the invention are useful, for instance, in specific bindingassays, for genotyping or phenotyping humans, affinity purification ofthe PCSK9 and in screening assays to identify other antagonists of PCSK9activity. Some of the ligands of the invention are useful for inhibitingbinding of PCSK9 to a congnate human receptor or protein, or inhibitingPCSK9-mediated activities.

The invention encompasses anti-PCSK9 (eg, PCSK9) antibody ligands havinga modified glycosylation pattern. In some applications, modification toremove undesirable glycosylation sites may be useful, or e.g., removalof a fucose moiety to increase antibody dependent cellular cytotoxicity(ADCC) function (see Shield et al. (2002) JBC 277:26733). In otherapplications, modification of galactosylation can be made in order tomodify complement dependent cytotoxicity (CDC).

In an example, the invention features a pharmaceutical compositioncomprising a ligand of the invention, wherein the ligand is or comprisesa recombinant human antibody or fragment thereof which specificallybinds the PCSK9 (eg, a rare variant as described herein) and apharmaceutically acceptable carrier. In one embodiment, the inventionfeatures a composition which is a combination of an antibody ligand orantigen-binding fragment of an antibody of the invention, and a secondtherapeutic agent. The second therapeutic agent may be any of ananti-inflammatory agent, an anti-angiogenesis agent, a painkiller, adiuretic, a chemotherapeutic agent, an anti-neoplastic agent, avasodilator, a vasoconstrictor, a statin, a beta blocker, a nutrient, anadjuvant, an anti-obesity agent and an anti-diabetes agent.

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the USA Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, including humans. A “pharmaceutically acceptable carrier,excipient, or adjuvant” refers to an carrier, excipient, or adjuvantthat can be administered to a subject, together with an agent, e.g., anyantibody or antibody chain described herein, and which does not destroythe pharmacological activity thereof and is nontoxic when administeredin doses sufficient to deliver a therapeutic amount of the agent.

In an example, the invention features a method for inhibiting PCSK9activity using the anti-PCSK9 ligand of the invention (eg, an antibodyor antigen-binding portion of the antibody of the invention), whereinthe therapeutic method comprises administering a therapeuticallyeffective amount of a pharmaceutical composition comprising the ligand.The disorder treated is any disease or condition which is improved,ameliorated, inhibited or prevented by removal, inhibition or reductionof PCSK9 activity.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

Genotyping & Phenotyping

The skilled person will be familiar with techniques that can be used foraccurate genotyping and application to the invention. These include thefollowing.

1 Hybridization-based methods1.1 Dynamic allele-specific hybridization1.2 Molecular beacons1.3 SNP microarrays2 Enzyme-based methods2.1 Restriction fragment length polymorphism2.2 PCR-based methods2.3 Flap endonuclease2.4 Primer extension2.5 5′-nuclease

2.6 Oligonucleotide Ligation Assay

3 Other post-amplification methods based on physical properties of DNA3.1 Single strand conformation polymorphism3.2 Temperature gradient gel electrophoresis3.3 Denaturing high performance liquid chromatography3.4 High-resolution melting of the entire amplicon3.5 Use of DNA mismatch-binding proteins3.6 SNPlex (SNPlex™ is a proprietary genotyping platform sold by AppliedBiosystems).

Next-generation sequencing technologies such as pyrosequencing is alsouseful.

Reference is also made to GB2444410A and the genotyping method disclosedtherein, which is incorporated herein by reference in its entirety.

Miniaturized assays, such as microarrays with oligonucleotide reagentsimmobilized on small surfaces, are frequently proposed for large-scalemutation analysis and high-throughput genotyping (Large-scaleidentification, mapping, and genotyping of single-nucleotidepolymorphisms in the human genome (Wang D G, Fan J B, Siao C J, Berno A,Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J,Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E,Mittmann M, Morris M S, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S,Hudson T J, Lipshutz R, Chee M, Lander E S, Science. 1998 May 15;280(5366):1077-82). Other high-throughput methods discriminate allelesby differential hybridization, primer extension, ligation and cleavageof an allele-specific probe (Review Accessing genetic variation:genotyping single nucleotide polymorphisms, Syvinen A C, Nat Rev Genet.2001 December; 2(12):930-42; Review Techniques patents for SNPgenotyping, Twyman R M, Primrose S B, Pharmacogenomics. 2003 January;4(1):67-79).

An approach for a fully automated, large-scale SNP analysis is the‘homogeneous’ assay, i.e. a single-phase assay without separation steps,permitting continual monitoring during amplification. The TaqMan™ assay(Applied Biosystems), originally designed for quantitative real-timePCR, is a homogeneous, single-step assay also used in determination ofmutation status of DNA (see, eg, A. A. Komar (ed.), Single NucleotidePolymorphisms, Methods in Molecular Biology 578, DOI10.1007/978-1-60327-411-1_19, Humana Press, a part of SpringerScience+Business Media, LLC; and Single Nucleotide Polymorphisms,Methods in Molecular Biology™ Volume 578, 2009, pp 293-306, The TaqManMethod for SNP Genotyping, Gong-Qing Shen et al). The TaqMan SNPGenotyping Assay exploits the 5′-exonuclease activity of AmpliTaq Gold™DNA polymerase to cleave a doubly labeled probe hybridized to theSNP-containing sequence of ssDNA. Cleavage separates a 5′-fluorophorefrom a 3′-quencher leading to detectable fluorescent signal. The use oftwo allele-specific probes carrying different fluorophores permits SNPdetermination in the same tube without any post-PCR processing. Genotypeis determined from the ratio of intensities of the two fluorescentprobes at the end of amplification. Thus, rather than taking advantageof the full set of real-time PCR data as in quantitative studies, onlyend-point data are used.

TaqMan SNP genotyping in a high-throughput, automated manner isfacilitated by the use of validated Pre-made TaqMan® Genotyping assays,but Custom TaqMan® Assays may also be used (High-throughput genotypingwith single nucleotide polymorphisms, Ranade K, Chang M S, Ting C T, PeiD, Hsiao C F, Olivier M, Pesich R, Hebert J, Chen Y D, Dzau V J, Curb D,Olshen R, Risch N, Cox D R, Botstein D, Genome Res. 2001 July;11(7):1262-8; Assessment of two flexible and compatible SNP genotypingplatforms: TaqMan SNP Genotyping Assays and the SNPlex GenotypingSystem, De la Vega F M, Lazaruk K D, Rhodes M D, Wenz M H, Mutat Res.2005 Jun. 3; 573(1-2):111-35). The results of the assay can beautomatically determined by genotyping software provided with real-timethermal cyclers (e.g. IQ software of Bio-Rad, Sequence DetectionSoftware of Applied Biosystems).

Single nucleotide polymorphisms (SNPs) can be determined using TaqMan™real-time PCR assays (Applied Biosystems) and commercial software thatassigns genotypes based on reporter probe signals at the end ofamplification. An algorithm for automatic genotype calling of SNPs usingthe full course of TaqMan real-time data is available for use (A.Callegaro et al, Nucleic Acids Res. 2006; 34(7): e56, Published online2006 April 14. doi: 10.1093/nar/gkl185, PMCID: PMC1440877). Thealgorithm is unique in that it classifies samples according to thebehavior of blanks (no DNA samples), which cluster with heterozygoussamples. This method of classification eliminates the need for positivecontrols and permits accurate genotyping even in the absence of agenotype class, for example when one allele is rare.

The skilled person will be familiar with techniques that can be used foraccurate phenotyping and application to the invention. These include theuse of amino acid sequencing of isolated target protein and comparisonof sequences from different variants (eg, with the most common variant).An antibody that specifically and selectively binds in the area of a SNPunder stringent conditions can also be used to identify a particularvariant. In another method, the genotype is determined and acorresponding amino acid sequence (phenotype) determined, eg, by insilico translation.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, or “reduction” are all used herein tomean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” typically means adecrease by at least 10% as compared to a reference level (e.g. theabsence of a given treatment) and can include, for example, a decreaseby at least about 10%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or more. As used herein, “reduction” doesnot encompass a complete reduction as compared to a reference level. Adecrease can be preferably down to a level accepted as within the rangeof normal for an individual without a given disorder. However, forexample, for the purposes of lowering or reducing cholesterol level, forexample, a reduction by about 5-10 points can be considered a “decrease”or “reduction.”

In certain aspects of all embodiments of the invention, the term“inhibition” is used. Inhibition refers and refers to decrease by atleast 10% as compared to a reference level (e.g. the absence of a giventreatment) and can include, for example, a decrease by at least about10%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or more including 100% inhibition as compared to areference level. “Complete inhibition” refers to a 100% inhibition ascompared to a reference level.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, an “increase” is a statistically significant increasein such level.

As used herein, the term “substantially” refers to the qualitativecondition of exhibiting total or near-total extent or degree of acharacteristic or property of interest. One of ordinary skill in thebiological arts will understand that biological and chemical phenomenararely, if ever, go to completion and/or proceed to completeness orachieve or avoid an absolute result. The term “substantially” istherefore used herein to capture the potential lack of completenessinherent in many biological and chemical phenomena. For the removal ofdoubt, “substantially” can refer to at least a 90% extent or degree of acharacteristic or property of interest, e.g. at least 90%, at least 92%,at least 95%, at least 98%, at least 99% or greater.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. In some embodiments, thesubject is a mammal, e.g., a primate, e.g., a human. The terms,“individual,” “patient” and “subject” are used interchangeably herein.In some embodiments, the subject can be a non-human vertebrate, e.g. aprimate, a rodent, a mouse, a rat, a pig, a sheep, a zebrafish, a frog,etc.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of adisease or condition, e.g., a cardiovascular condition. A subject can bemale or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatmentor one or more complications related to such a condition, andoptionally, have already undergone treatment for the condition or theone or more complications related to the condition. Alternatively, asubject can also be one who has not been previously diagnosed as havingthe condition or one or more complications related to the condition. Forexample, a subject can be one who exhibits one or more risk factors forthe condition or one or more complications related to the condition or asubject who does not exhibit risk factors.

A “subject in need” or “human in need” of treatment for a particularcondition can be a subject having that condition, such as increasedcholesterol levels, diagnosed as having that condition, or at risk ofdeveloping that condition.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids with natural aminoacids. When referring to “modified polypeptides” one refers topolypeptides that include modified amino acids (e.g., phosphorylated,glycated, glycosylated, etc.) and amino acid analogs, regardless of itssize or function. “Protein” and “polypeptide” are often used inreference to relatively large polypeptides, whereas the term “peptide”is often used in reference to small polypeptides, but usage of theseterms in the art overlaps. The terms “protein” and “polypeptide” areused interchangeably herein when referring to a gene product andfragments thereof. Thus, exemplary polypeptides or proteins include geneproducts, naturally occurring proteins with the specified sequence. Onecan also use peptide homologs, peptide orthologs, peptide paralogs,peptide fragments and other equivalents, variants, fragments, andanalogs of the peptides as these terms are understood by one of ordinaryskill in the art.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid. The nucleic acid canbe either single-stranded or double-stranded. A single-stranded nucleicacid can be one nucleic acid strand of a denatured double-stranded DNA.Alternatively, it can be a single-stranded nucleic acid not derived fromany double-stranded DNA. In one aspect, the nucleic acid can be DNA. Inanother aspect, the nucleic acid can be RNA. Suitable nucleic acidmolecules are DNA, including genomic DNA or cDNA. Other suitable nucleicacid molecules are RNA, including mRNA. In some aspects one can also useanalogs of nucleic acids.

As used herein, the term “nucleic acid probe” refers to an isolatedoligonucleotide molecule having a nucleic acid sequence which canhybridize to a target nucleic acid sequence, e.g. specifically hybridizeto the target sequence. In some embodiments, a nucleic acid probe canfurther comprise a detectable label. In some embodiments, a nucleic acidprobe can be attached to a solid surface. In some embodiments, a nucleicacid from is from about 5 nt to about 100 nt in length.

As used herein, the term “siRNA” refers to a nucleic acid that forms anRNA molecule comprising two individual strands of RNA which aresubstantially complementary to each other. Typically, the siRNA is atleast about 15-40 nucleotides in length (e.g., each complementarysequence of the double stranded siRNA is about 15-40 nucleotides inlength, and the double stranded siRNA is about 15-40 base pairs inlength, preferably about 19-25 base nucleotides, e.g., 19, 20, 21, 22,23, 24, or 25 nucleotides in length). In some embodiments, a siRNA canbe blunt-ended. In some embodiments, a siRNA can comprise a 3′ and/or 5′overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5nucleotides. The length of the overhang is independent between the twostrands, i.e., the length of the overhang on one strand is not dependenton the length of the overhang on the second strand. The siRNA moleculescan also comprise a 3′ hydroxyl group. In some embodiments, the siRNAcan comprise a 5′ phosphate group. A siRNA has the ability to reduce orinhibit expression of a gene or target RNA when the siRNA is present orexpressed in the same cell as the target gene, e.g. the target RNA.siRNA-dependent post-transcriptional silencing of gene expressioninvolves cutting the target RNA molecule at a site guided by the siRNA.

As used herein, “PCSK9” or “proprotein convertase subtilisin/kexin type9” refers to a serine protease involved in regulating the levels of thelow density lipoprotein receptor (LDLR) protein (Horton et al., 2007;Seidah and Prat, 2007). PCSK9 has been shown to directly interact withthe LDLR protein, be endocytosed along with the LDLR, andco-immunofluoresce with the LDLR throughout the endosomal pathway(Lagace et al., 2006). PCSK9 is a prohormone-proprotein convertase inthe subtilisin (S8) family of serine proteases (Seidah et al., 2003).The sequence of PCSK9 for a variety of species is known, e.g., humanPCSK9 (NCBI Gene ID No: 255738). Nucleotide and polypeptide sequencesfor a number of PCSK9 isoforms are provided herein, e.g., SEQ ID NOs:1-37.

PCSK9 exists as both a pro-form and a mature form. Autocatalysis of thePCSK9 proform occurs between Gln152 and Ser153 (VFAQ|SIP) (Naureckieneet al., 2003), and has been shown to be required for its secretion fromcells (Seidah et al., 2003). The inactive form prior to this cleavagecan be referred to herein as the “inactive”, “pro-form”, or“unprocessed” form of PCSK9. The C-terminal fragment generated by theautocatalysis event can be referred to herein as the “mature,”“cleaved”, “processed” or “active” PCSK9. Examples of pro-form andmature PCSK9 isoforms are provided herein, see, e.g. SEQ ID NOs: 1-27.

As used herein, the “catalytic domain” of PCSK9 refers to the portion ofa PCSK9 polypeptide corresponding to positions 153 to 449 of PCSK9, e.g.of SEQ ID NO: 1. As used herein, the “C-terminal domain” of PCSK9 refersto the portion of a PCSK9 polypeptide corresponding to positions 450-692of PCSK9, e.g., of SEQ ID NO: 1.

As used herein, a disease or condition “mediated by PCSK9” refers to adisease or condition which is caused by or characterized by a change inPCSK9, e.g. a change in expression level, a change in activity, and/orthe presence of a variant or mutation of PCSK9. Non-limiting examples ofsuch diseases or conditions can include, for example, a lipid disorder,hyperlipoproteinemia, hyperlipidemia; dyslipidemia;hypercholesterolemia, a heart attack, a stroke, coronary heart disease,atherosclerosis, peripheral vascular disease, claudication, type IIdiabetes, high blood pressure, and a cardiovascular disease orcondition. In an example, the disease or condition is an inflammatory orautoimmune disease or condition. Methods of identifying and/ordiagnosing such diseases and conditions are well known to medicalpractitioners of ordinary skill.

A subject at risk of having or developing a disease or conditionmediated by PCSK9 can be a subject exhibiting one or more signs orsymptoms of such a disease or condition or having one or more riskfactors for such a disease or condition, e.g. being overweight, havingelevated cholesterol level, comprising one or more genetic polymorphismsknown to predispose to the disease or condition, e.g., elevatedcholesterol level, such as having a mutation in the LDLR (encodinglow-density lipoprotein receptor) or APOB (encoding apolipoprotein B) orin the PCSK9 gene and/or having a family history of such a disease orcondition.

As used herein, “ligand” refers to a molecule which can bind, e.g.,specifically bind, to a second molecule or receptor. In someembodiments, a ligand can be, e.g., an antibody, antibody fragment,antibody portion, and/or affibody.

The term “variant” as used herein refers to a peptide or nucleic acidthat differs from the polypeptide or nucleic acid (eg, the most commonone in humans, eg, most frequent in a database as disclosed herein, suchas the 1000 Genomes Project database) by one or more amino acid ornucleic acid deletions, additions, yet retains one or more specificfunctions or biological activities of the naturally occurring molecule.Amino acid substitutions include alterations in which an amino acid isreplaced with a different naturally-occurring amino acid residue. Suchsubstitutions may be classified as “conservative”, in which case anamino acid residue contained in a polypeptide is replaced with anothernaturally occurring amino acid of similar character either in relationto polarity, side chain functionality or size. Such conservativesubstitutions are well known in the art. Substitutions encompassed bythe present invention may also be “non-conservative”, in which an aminoacid residue which is present in a peptide is substituted with an aminoacid having different properties, such as naturally-occurring amino acidfrom a different group (e.g., substituting a charged or hydrophobicamino; acid with alanine), or alternatively, in which anaturally-occurring amino acid is substituted with a non-conventionalamino acid. In some embodiments amino acid substitutions areconservative. Also encompassed within the term variant when used withreference to a polynucleotide or polypeptide, refers to a polynucleotideor polypeptide that can vary in primary, secondary, or tertiarystructure, as compared to a reference polynucleotide or polypeptide,respectively (e.g., as compared to a wild-type polynucleotide orpolypeptide).

Variants of PCSK9 are provided elsewhere herein. Variants of PCSK9 caninclude the forms described herein as a, f, c, r, p, m, e h, aj, and q.Sequences of these variants are provided herein, see, e.g, SEQ IDNOs:1-27 and in Table 1.

In some aspects, one can use “synthetic variants”, “recombinantvariants”, or “chemically modified” polynucleotide variants orpolypeptide variants isolated or generated using methods well known inthe art. “Modified variants” can include conservative ornon-conservative amino acid changes, as described below. Polynucleotidechanges can result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence. Some aspects use include insertion variants, deletion variantsor substituted variants with substitutions of amino acids, includinginsertions and substitutions of amino acids and other molecules) that donot normally occur in the peptide sequence that is the basis of thevariant, for example but not limited to insertion of ornithine which donot normally occur in human proteins. The term “conservativesubstitution,” when describing a polypeptide, refers to a change in theamino acid composition of the polypeptide that does not substantiallyalter the polypeptide's activity. For example, a conservativesubstitution refers to substituting an amino acid residue for adifferent amino acid residue that has similar chemical properties.Conservative amino acid substitutions include replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, or athreonine with a serine.

“Conservative amino acid substitutions” result from replacing one aminoacid with another having similar structural and/or chemical properties,such as the replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, or a threonine with a serine. Thus, a“conservative substitution” of a particular amino acid sequence refersto substitution of those amino acids that are not critical forpolypeptide activity or substitution of amino acids with other aminoacids having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, etc.) such that the substitutionof even critical amino acids does not reduce the activity of thepeptide, (i.e. the ability of the peptide to penetrate the blood brainbarrier (BBB)). Conservative substitution tables providing functionallysimilar amino acids are well known in the art. For example, thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company(1984), incorporated by reference in its entirety.) In some embodiments,individual substitutions, deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids can alsobe considered “conservative substitutions” if the change does not reducethe activity of the peptide. Insertions or deletions are typically inthe range of about 1 to 5 amino acids. The choice of conservative aminoacids may be selected based on the location of the amino acid to besubstituted in the peptide, for example if the amino acid is on theexterior of the peptide and expose to solvents, or on the interior andnot exposed to solvents.

In alternative embodiments, one can select the amino acid which willsubstitute an existing amino acid based on the location of the existingamino acid, i.e. its exposure to solvents (i.e. if the amino acid isexposed to solvents or is present on the outer surface of the peptide orpolypeptide as compared to internally localized amino acids not exposedto solvents). Selection of such conservative amino acid substitutionsare well known in the art, for example as disclosed in Dordo et al, J.MoI Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol.119(1986); 205-218 and S. French and B. Robson, J. MoI. Evol. 19(1983)171. Accordingly, one can select conservative amino acid substitutionssuitable for amino acids on the exterior of a protein or peptide (i.e.amino acids exposed to a solvent), for example, but not limited to, thefollowing substitutions can be used: substitution of Y with F, T with Sor K, P with A, E with D or Q, N with D or G, R with K, G with N or A, Twith S or K, D with N or E, I with L or V, F with Y, S with T or A, Rwith K, G with N or A, K with R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acidsubstitutions encompassed suitable for amino acids on the interior of aprotein or peptide, for example one can use suitable conservativesubstitutions for amino acids is on the interior of a protein or peptide(i.e. the amino acids are not exposed to a solvent), for example but notlimited to, one can use the following conservative substitutions: whereY is substituted with F, T with A or S, I with L or V, W with Y, M withL, N with D, G with A, T with A or S, D with N, I with L or V, F with Yor L, S with A or T and A with S, G, T or V. In some embodiments,non-conservative amino acid substitutions are also encompassed withinthe term of variants.

As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgEmolecules or antigen-specific antibody fragments thereof (including, butnot limited to, a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, singledomain antibody, closed conformation multispecific antibody,disulphide-linked scfv, diabody), whether derived from any species thatnaturally produces an antibody, or created by recombinant DNAtechnology; whether isolated from serum, B-cells, hybridomas,transfectomas, yeast or bacteria. Antibodies can be humanized usingroutine technology.

As described herein, an “antigen” is a molecule that is bound by abinding site on an antibody agent. Typically, antigens are bound byantibody ligands and are capable of raising an antibody response invivo. An antigen can be a polypeptide, protein, nucleic acid or othermolecule or portion thereof. The term “antigenic determinant” refers toan epitope on the antigen recognized by an antigen-binding molecule, andmore particularly, by the antigen-binding site of said molecule.

As used herein, the term “antibody fragment” refers to a polypeptidethat includes at least one immunoglobulin variable domain orimmunoglobulin variable domain sequence and which specifically binds agiven antigen. An antibody fragment can comprise an antibody or apolypeptide comprising an antigen-binding domain of an antibody. In someembodiments, an antibody fragment can comprise a monoclonal antibody ora polypeptide comprising an antigen-binding domain of a monoclonalantibody. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as VH), and a light (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody fragment” encompassesantigen-binding fragments of antibodies (e.g., single chain antibodies,Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, anddomain antibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J.Immunol. 1996; 26(3):629-39; which is incorporated by reference hereinin its entirety)) as well as complete antibodies. An antibody can havethe structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypesand combinations thereof). Antibodies can be from any source, includingmouse, rabbit, pig, rat, and primate (human and non-human primate) andprimatized antibodies. Antibodies also include midibodies, humanizedantibodies, chimeric antibodies, and the like.

As used herein, “antibody variable domain” refers to the portions of thelight and heavy chains of antibody molecules that include amino acidsequences of Complementarity Determining Regions (CDRs; ie., CDR1, CDR2,and CDR3), and Framework Regions (FRs). VH refers to the variable domainof the heavy chain. VL refers to the variable domain of the light chain.According to the methods used in this invention, the amino acidpositions assigned to CDRs and FRs may be defined according to Kabat(Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991)) or according to IMGTnomenclature.

D domain or region refers to the diversity domain or region of anantibody chain. J domain or region refers to the joining domain orregion of an antibody chain.

An antibody “gene segment”, e.g. a VH gene segment, D gene segment, orJH gene segment refers to oligonucleotide having a nucleic acid sequencethat encodes that portion of an antibody, e.g. a VH gene segment is anoligonucleotide comprising a nucleic acid sequence that encodes apolypeptide VH domain.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRs has beenprecisely defined (see, IMGT or Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated byreference herein in their entireties). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The terms “antigen-binding fragment” or “antigen-binding domain”, whichare used interchangeably herein are used to refer to one or morefragments of a full length antibody that retain the ability tospecifically bind to a target of interest. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of a full lengthantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment including two Fab fragments linked by a disulfide bridge at thehinge region; (iii) an Fd fragment consisting of the VH and CH1 domains;(iv) an Fv fragment consisting of the VL and VH domains of a single armof an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544-546; which is incorporated by reference herein in its entirety),which consists of a VH or VL domain; and (vi) an isolatedcomplementarity determining region (CDR) that retains specificantigen-binding functionality.

As used herein, the term “antibody binding site” refers to a polypeptideor domain that comprises one or more CDRs of an antibody and is capableof binding an antigen. For example, the polypeptide comprises a CDR3(eg, HCDR3). For example the polypeptide comprises CDRs 1 and 2 (eg,HCDR1 and 2) or CDRs 1-3 of a variable domain of an antibody (eg,HCDRs1-3). In an example, the antibody binding site is provided by asingle variable domain (eg, a VH or VL domain). In another example, thebinding site comprises a VH/VL pair or two or more of such pairs.

As used herein, the term “specific binding” refers to a chemicalinteraction between two molecules, compounds, cells and/or particleswherein the first entity binds to the second, target entity with greaterspecificity and affinity than it binds to a third entity which is anon-target. For example, in an diagnostic test the specific binding of aligand can distinguish between two variant PCSK9 proteins as describedherein. In some embodiments, specific binding can refer to an affinityof the first entity for the second target entity which is at least 10times, at least 50 times, at least 100 times, at least 500 times, atleast 1000 times or greater than the affinity for the third nontargetentity. In the context of oligonucleotide strands which interact viahybridization, specific binding can be “specific hybridization.”

Additionally, and as described herein, a recombinant human(ized)antibody can be further optimized to decrease potential immunogenicity,while maintaining functional activity, for therapy in humans. In thisregard, functional activity means a polypeptide capable of displayingone or more known functional activities associated with a recombinantantibody or antibody reagent thereof as described herein. Suchfunctional activities include, e.g. the ability to bind to a targetmolecule.

The term “immunizing” refers to the step or steps of administering oneor more antigens to an animal so that antibodies can be raised in theanimal Generally, immunizing comprises injecting the antigen or antigensinto the animal. Immunization can involve one or more administrations ofthe antigen or antigens. Suitable methods are prime-boost and RIMMSprocedures as known to the skilled person in the art.

As used herein, an “affibody” refers to a relatively small syntheticprotein molecule that has high binding affinity for a target protein(e.g. for PCSK9 or a variant thereof). Affibodies are composed of athree-helix bundle domain derived from the IgG-binding domain ofstaphylococcal protein A. The protein domain consists of a 58 amino acidsequence, with 13 randomized amino acids affording a range of affibodyvariants. Despite being significantly smaller than an antibody (anaffibody weighs about 6 kDa while an antibody commonly weighs about 150kDa), an affibody molecule works like an antibody since its binding siteis approximately equivalent in surface area to the binding site of anantibody.

As used herein, “VH3-23*04” refers to a human VH domain variantcomprising the polypeptide sequence of SEQ ID NO: 38. As opposed to thereference sequence, VH3-23*04 has a valine residue instead of a leucineresidue (see FIGS. 3 and 4 ; L24V, numbering including signal sequence;valine at position 5 shown in FIG. 4 ) as a result of the presence ofthe rs56069819 SNP in the nucleic acid sequence encoding the VH domain.As used herein, “rs56069819” refers to a mutation or variant in a VHgene segment from adenosine to cytosine (or thymine to guanine,depending upon the strand of DNA which is being read), resulting in theVH domain encoding VH3-23*04. Rs56069819 is depicted in FIG. 4 and SEQID NO: 39, which demonstrate the T->G mutation (it is noted that thedbSNP entry for RS5606819 depicts the other strand, which comprises theA->C mutation). Further description of VH3-23*04 can be found, e.g., inUS Patent Publication 2013/0071405; which is incorporated by referenceherein in its entirety.

As used herein, “determine” or “determining” refers to ascertaining,e.g., by a quantitative or qualitative analysis. As used herein, “hasbeen determined” can refer to ascertaining on the basis of previouslyobtained information or simultaneously obtained information.

In some aspects of all embodiments of the invention selecting caninclude automation such as a computer implemented software program thatupon input of the relevant data such as ethnicity or a panel of SNP datacan make the determination based on the instructions set forth herein.

As used herein, “assaying” refers to assessing, evaluating, quantifying,measuring, or characterizing an analyte, e.g., measuring the level of ananalyte in a sample, identifying an analyte, or detecting the presenceor absence of an analyte in a sample. In some embodiments, assayingrefers to detecting a presence or absence of the analyte of interest. Insome embodiments, assaying refers to quantifying an amount of ananalyte, e.g., providing a measure of concentration or degree of analyteabundance. In some embodiments, assaying refers to enumerating thenumber of molecules of analyte present in a sample and/or specimen,e.g., to determine an analyte copy number.

As used herein “multiplex” refers to the carrying out of a method orprocess simultaneously and in the same reaction vessel on two or more,typically three or more, different target sequences, e.g. on two or moreisoforms of PCSK9, or PCSK9 and an additional target. A multiplexanalysis typically includes analysis of 10-50; 10-100; 10-1000, 10-5000,10-10000 reactions in a multiplex format, such as a multiwall, an array,or a multichannel reaction.

Often the analysis or multiplex analysis is also automated usingrobotics and typically software executed by a computer and may include arobotic handling of samples, automatic or robotic selection of positiveor negative results, assaying for presence of absence of a target, suchas a nucleic acid polymorphism or a protein variant.

The term “biological sample” or “test sample” as used herein denotes asample taken or isolated from a biological organism, e.g., a sample froma subject. Exemplary biological samples include, but are not limited to,a biofluid sample; serum; plasma; urine; saliva; hair, epithelial cells,skin, a tumor biopsy and/or tissue sample etc. The term also includes amixture of the above-mentioned samples. The term “test sample” or“biological sample” also includes untreated or pretreated (orpre-processed) biological samples. For the analysis of nucleic acids,the biological sample should typically comprise at least one cellcomprising nucleic acids.

The test sample can be obtained by removing a sample of cells from asubject, but can also be accomplished by using previously isolated cells(e.g. isolated at a prior time point and isolated by the same or anotherperson). In addition, the test sample can be freshly collected or apreviously collected, refrigerated, frozen or otherwise preservedsample.

In some embodiments, the test sample can be an untreated test sample. Asused herein, the phrase “untreated test sample” refers to a test samplethat has not had any prior sample pre-treatment except for dilutionand/or suspension in a solution. Exemplary methods for treating a testsample include, but are not limited to, centrifugation, filtration,sonication, homogenization, heating, freezing and thawing, andcombinations thereof. In some embodiments, the test sample can be afrozen test sample, e.g., a frozen tissue. The frozen sample can bethawed before employing methods, assays and systems described herein.After thawing, a frozen sample can be centrifuged before being subjectedto methods, assays and systems described herein. In some embodiments,the test sample is a clarified test sample, for example, bycentrifugation and collection of a supernatant comprising the clarifiedtest sample. In some embodiments, a test sample can be a pre-processedtest sample, for example, supernatant or filtrate resulting from atreatment selected from the group consisting of centrifugation,filtration, thawing, purification, and any combinations thereof. In someembodiments, the test sample can be treated with a chemical and/orbiological reagent. Chemical and/or biological reagents can be employedto protect and/or maintain the stability of the sample, includingbiomolecules (e.g., nucleic acid and protein) therein, duringprocessing. One exemplary reagent is a protease inhibitor, which isgenerally used to protect or maintain the stability of protein duringprocessing. The skilled artisan is well aware of methods and processesappropriate for pre-processing of biological samples required fordetermination of the level of an expression product as described herein.

As used herein, “genotyping” refers to a process of determining thespecific allelic composition of a cell and/or subject at one or moreposition within the genome, e.g. by determining the nucleic acidsequence at that position. Genotyping refers to a nucleic acid analysisand/or analysis at the nucleic acid level. As used herein, “phenotyping”refers a process of determining the identity and/or composition of anexpression product of a cell and/or subject, e.g. by determining thepolypeptide sequence of an expression product. Phenotyping refers to aprotein analysis and/or analysis at the protein level.

As used herein, the term “nucleic acid amplification” refers to theproduction of additional copies of a nucleic acid sequence and istypically carried out using polymerase chain reaction (PCR) or ligasechain reaction (LCR) technologies well known in the art (Dieffenbach, C.W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, ColdSpring Harbor Press, Plainview, N.Y.). Other methods for amplificationare also contemplated in aspects of the invention.

The term “allele-specific amplification” refers to a reaction (e.g., PCRreaction) in which at least one of the primers (e.g., allele-specificprimer) is chosen from a polymorphic area of gene (e.g., singlenucleotide polymorphism), with the polymorphism located at or near theprimer's 3′-end. A mismatched primer will not initiate amplification,whereas a matched primer will initiate amplification. The appearance ofan amplification product is indicative of the presence of thepolymorphism.

As used herein, “sequencing” refers to the determination of the exactorder of nucleotide bases in a strand of DNA (deoxyribonucleic acid) orRNA (ribonucleic acid) or the exact order of amino acids residues orpeptides in a protein. Nucleic acid sequencing can be done using Sangersequencing or next-generation high-throughput sequencing.

As used herein “next-generation sequencing” refers to oligonucleotidesequencing technologies that have the capacity to sequenceoligonucleotides at speeds above those possible with conventionalsequencing methods (e.g. Sanger sequencing), due to performing andreading out thousands to millions of sequencing reactions in parallel.Non-limiting examples of next-generation sequencing methods/platformsinclude Massively Parallel Signature Sequencing (Lynx Therapeutics); 454pyro-sequencing (454 Life Sciences/Roche Diagnostics); solid-phase,reversible dye-terminator sequencing (Solexa/Illumina): SOLiD technology(Applied Biosystems); Ion semiconductor sequencing (ION Torrent); DNAnanoball sequencing (Complete Genomics); and technologies available fromPacific Biosciences, Intelligen Bio-systems, Oxford NanoporeTechnologies, and Helicos Biosciences. Next-generation sequencingtechnologies and the constraints and design parameters of associatedsequencing primers are well known in the art (see, e.g. Shendure, etal., “Next-generation DNA sequencing,” Nature, 2008, vol. 26, No. 10,1135-1145; Mardis, “The impact of next-generation sequencing technologyon genetics,” Trends in Genetics, 2007, vol. 24, No. 3, pp. 133-141; Su,et al., “Next-generation sequencing and its applications in moleculardiagnostics” Expert Rev Mol Diagn, 2011, 11(3):333-43; Zhang et al.,“The impact of next-generation sequencing on genomics”, J GenetGenomics, 2011, 38(3):95-109; (Nyren, P. et al. Anal Biochem 208: 17175(1993); Bentley, D. R. Curr Opin Genet Dev 16:545-52 (2006); Strausberg,R. L., et al. Drug Disc Today 13:569-77 (2008); U.S. Pat. Nos.7,282,337; 7,279,563; 7,226,720; 7,220,549; 7,169,560; 6,818,395;6,911,345; US Pub. Nos. 2006/0252077; 2007/0070349; and 20070070349;which are incorporated by reference herein in their entireties).

As used herein, “nucleic acid hybridization” refers to the pairing ofcomplementary RNA and DNA strands as well as the pairing ofcomplementary DNA single strands. In some embodiments, nucleic acidhybridization can refer to a method of determining a nucleic acidsequence and/or identity by hybridizing a nucleic acid sample with aprobe, e.g. Northern or Southern blot analysis or microarray analysis.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorder.Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced. Alternatively, treatment is “effective” if theprogression of a disease is reduced or halted. That is, “treatment”includes not just the improvement of symptoms or markers, but also acessation of, or at least slowing of, progress or worsening of symptomscompared to what would be expected in the absence of treatment.Beneficial or desired clinical results include, but are not limited to,alleviation of one or more symptom(s), diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, remission (whether partial or total), and/or decreasedmortality, whether detectable or undetectable. The term “treatment” of adisease also includes providing relief from the symptoms or side-effectsof the disease (including palliative treatment). For treatment to beeffective a complete cure is not contemplated. The method can in certainaspects include cure as well.

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

Multiple compositions can be administered separately or simultaneously.Separate administration refers to the two compositions beingadministered at different times, e.g. at least 10, 20, 30, or 10-60minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. One canalso administer compositions at 24 hours apart, or even longer apart.Alternatively, two or more compositions can be administeredsimultaneously, e.g. less than 10 or less than 5 minutes apart.Compositions administered simultaneously can, in some aspects, beadministered as a mixture, with or without similar or different timerelease mechanism for each of the components.

As used herein, “contacting” refers to any suitable means fordelivering, or exposing, an agent to at least one complex, enzyme, orcell. Exemplary delivery methods include, but are not limited to, directdelivery to cell culture medium, perfusion, injection, or other deliverymethod well known to one skilled in the art.

As used herein, “obtain” refers to any method of acquiring, securing,procuring, or coming into the possession of, e.g. a sample. Obtaining abiological sample from a subject can comprise physical removing a samplefrom a subject (e.g. drawing blood or taking a hair or saliva sample)without or without active participation from the subject; receiving asample from a subject (e.g. the subject collects a saliva or hair samplethemselves and provides it, e.g. in a container provided for thepurpose); or procuring a sample from a storage facility, medicalfacility, or medical provider. Obtain from the human or subject, refersto an active step of, e.g., drawing blood or taking a tissue or cellsample.

As used herein, “cholesterol level” refers to a level of one or more oftotal cholesterol, LDL cholesterol, HDL cholesterol, and/ortriglycerides. Cholesterol levels can be the level of cholesterol in theblood of a subject.

As used herein in reference to cholesterol levels, “maintain” refers topreventing the level from worsening (e.g. increasing). In someembodiments, maintaining a particular level refers to a process thatresults in the cholesterol level not increasing by more than 10% overtime. Maintaining may also refer to maintaining a previously achievedlevel. For example, if a human has received statin treatment, one canmaintain the cholesterol level achieved using the statin treatment.

In some embodiments, the subject treated according to the methodsdescribed herein has previously had their cholesterol level reduced. Asused herein, “previously reduced” indicates that at a prior point intime, the subject experienced a decrease in cholesterol levels. Thedecrease can be due to administration of a pharmaceutical composition(e.g. administration of a composition as described herein or anothercomposition, e.g. a statin) or due to another cause, e.g. a change indiet and/or exercise.

An existing treatment for high cholesterol levels is the administrationof a statin. As referred to herein, a “statin” (also known as HMG-CoAreductase inhibitors) are inhibitors of the enzyme HMG-coA reductase,which mediates cholesterol production in the liver. Statins, bycompetitively binding HMG-CoA reductase, prevent the binding of HMG-CoAto the enzyme and thereby inhibit the activity of the reductase (e.g.the production of mevalonate). Non-limiting examples of statins caninclude atorvastatin (LIPITOR™), fluvastatin (LESCOL™), lovastatin(MEVACOR™, ALTOCOR™), pitavastatin (LIVALO™), pravastatin (PRAVACHOL™),rosuvastatin (CRESTOR™), and simvastatin (ZOCOR™). Statins can beadministered in combination with other agents, e.g. the combination ofezetimibe and simvastatin.

Some subjects are, or become, resistant to statin treatment. As usedherein, “resistant to statin treatment” or “reduced responsiveness tostatin treatment” refers to a subject exhibiting a statisticallysignificantly lower response to the administration of a statin ascompared to a reference level. The reference level can be, e.g., theaverage response for a population of subjects or the level of theindividual subject at an earlier date. A response to statin treatment isreadily measured by one of skill in the art, e.g., measurement ofcholesterol levels, changes in cholesterol levels, and/or HMG-CoAreductase activity.

As used herein, the term “detectable label” refers to a molecule ormoiety that can be detected, e.g. measured and/or determined to bepresent or absent. Detectable labels can comprise, for example, alight-absorbing dye, a fluorescent dye, or a radioactive label.Detectable labels, methods of detecting them, and methods ofincorporating them into reagents (e.g. antibodies and nucleic acidprobes) are well known in the art.

In some embodiments, detectable labels can include labels that can bedetected by spectroscopic, photochemical, biochemical, immunochemical,electromagnetic, radiochemical, or chemical means, such as fluorescence,chemifluoresence, or chemiluminescence, or any other appropriate means.The detectable labels used in the methods described herein can beprimary labels (where the label comprises a moiety that is directlydetectable or that produces a directly detectable moiety) or secondarylabels (where the detectable label binds to another moiety to produce adetectable signal, e.g., as is common in immunological labeling usingsecondary and tertiary antibodies). The detectable label can be linkedby covalent or non-covalent means to the reagent. Alternatively, adetectable label can be linked such as by directly labeling a moleculethat achieves binding to the reagent via a ligand-receptor binding pairarrangement or other such specific recognition molecules. Detectablelabels can include, but are not limited to radioisotopes, bioluminescentcompounds, chromophores, antibodies, chemiluminescent compounds,fluorescent compounds, metal chelates, and enzymes.

In other embodiments, the detectable label can be a fluorescentcompound. When the fluorescently label is exposed to light of the properwavelength, its presence can then be detected due to fluorescence. Insome embodiments, a detectable label can be a fluorescent dye molecule,or fluorophore including, but not limited to fluorescein, phycoerythrin,phycocyanin, o-phthaldehyde, fluorescamine, Cy3™, Cy5™,allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandemconjugates such as phycoerythrin-Cy5™, green fluorescent protein,rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™,rhodamine and derivatives (e.g., Texas red and tetrarhodimineisothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™,6-carboxyfhiorescein (commonly known by the abbreviations FAM and F),6-carboxy-2′,4′,7′,4,7-hexachlorofiuorescein (HEX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfiuorescein (JOE or J),N,N,N′,N′-tetramethyl-6carboxyrhodamine (TAMRA or T),6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5),6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes,e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimidedyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidiumdyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes;polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyesand quinoline dyes. In some embodiments, a detectable label can be aradiolabel including, but not limited to ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, and³³P. In some embodiments, a detectable label can be an enzyme including,but not limited to horseradish peroxidase and alkaline phosphatase. Anenzymatic label can produce, for example, a chemiluminescent signal, acolor signal, or a fluorescent signal. Enzymes contemplated for use as adetectable label can include, but are not limited to, malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase,asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease,catalase, glucose-VI-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. In some embodiments, a detectable label is achemiluminescent label, including, but not limited to lucigenin,luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole,acridinium salt and oxalate ester. In some embodiments, a detectablelabel can be a spectral colorimetric label including, but not limited tocolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, and latex) beads.

In some embodiments, reagents can also be labeled with a detectable tag,such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detectionsystems can also be used, for example, a biotin-streptavidin system. Inthis system, the antibodies immunoreactive (i. e. specific for) with thebiomarker of interest is biotinylated. Quantity of biotinylated antibodybound to the biomarker is determined using a streptavidin-peroxidaseconjugate and a chromagenic substrate. Such streptavidin peroxidasedetection kits are commercially available, e. g. from DAKO; Carpinteria,Calif. A reagent can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the reagent using such metal chelating groupsas diethylenetriaminepentaacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

As used herein, “authorization number” or “marketing authorizationnumber” refers to a number issued by a regulatory agency upon thatagency determining that a particular medical product and/or compositionmay be marketed and/or offered for sale in the area under the agency'sjurisdiction. As used herein “regulatory agency” refers to one of theagencies responsible for evaluating, e.g, the safety and efficacy of amedical product and/or composition and controlling the sales/marketingof such products and/or compositions in a given area. The Food and DrugAdministration (FDA) in the US and the European Medicines Agency (EPA)in Europe are but two examples of such regulatory agencies. Othernon-limiting examples can include SDA, MPA, MHPRA, IMA, ANMAT, Hong KongDepartment of Health-Drug Office, CDSCO, Medsafe, and KFDA.

As used herein, “injection device” refers to a device that is designedfor carrying out injections, an injection including the steps oftemporarily fluidically coupling the injection device to a person'stissue, typically the subcutaneous tissue. An injection further includesadministering an amount of liquid drug into the tissue and decoupling orremoving the injection device from the tissue. In some embodiments, aninjection device can be an intravenous device or IV device, which is atype of injection device used when the target tissue is the blood withinthe circulatory system, e.g., the blood in a vein. A common, butnon-limiting example of an injection device is a needle and syringe.

As used herein, a “buffer” refers to a chemical agent that is able toabsorb a certain quantity of acid or base without undergoing a strongvariation in pH.

As used herein, “packaging” refers to how the components are organizedand/or restrained into a unit fit for distribution and/or use. Packagingcan include, e.g., boxes, bags, syringes, ampoules, vials, tubes,clamshell packaging, barriers and/or containers to maintain sterility,labeling, etc.

As used herein, “instructions” refers to a display of written, printedor graphic matter on the immediate container of an article, for examplethe written material displayed on a vial containing a pharmaceuticallyactive agent, or details on the composition and use of a product ofinterest included in a kit containing a composition of interest.Instructions set forth the method of the treatment as contemplated to beadministered or performed.

As used herein, a “solid surface” refers to an object suitable for theattachment of biomolecules. Non-limiting examples of a solid surface caninclude a particle (including, but not limited to an agarose or latexbead or particle or a magnetic particle), a bead, a nanoparticle, apolymer, a substrate, a slide, a coverslip, a plate, a dish, a well, amembrane, and/or a grating. The solid surface can include many differentmaterials including, but not limited to, polymers, plastics, resins,polysaccharides, silicon or silica based materials, carbon, metals,inorganic glasses, and membranes.

As used herein, “classification” of a subject, e.g., classification ofthe subject's ancestry refers to determining if the subject hasbiological ancestors who originated in a particular geographical area,and are therefore likely to have particular genetic variants found inthe populations which have historically occupied that area.Classification can comprise, e.g. obtaining information on the subject'sfamily, interviewing the subject or a family member regarding theirbiological family's ancestry, and/or genetic testing. Classification canbe on the basis used for the 1000 Genomes Project, as will be familiarto the skilled person in the art. In some embodiments, the subject canbe classified as being of a particular ancestry if at least thesubject's genome comprises a substantial number of different alleles incommon with other humans of that ancestry (eg, determined by referenceto the 1000 Genomes Project database), for example, at least 10, 20, 30,40, 50 or 100 or more alleles in common. Abbreviations for particularancestral groups are provided in Table 3.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean 1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); BenjaminLewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009,Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds.,Academic Press Inc., San Diego, USA (1987); Current Protocols in ProteinScience (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: AManual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998) which are all incorporated by reference herein in theirentireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

It will be understood that particular configurations, aspects, examples,clauses and embodiments described herein are shown by way ofillustration and not as limitations of the invention. The principalfeatures of this invention can be employed in various embodimentswithout departing from the scope of the invention. Those skilled in theart will recognize, or be able to ascertain using no more than routinestudy, numerous equivalents to the specific procedures described herein.Such equivalents are considered to be within the scope of this inventionand are covered by the claims. All publications and patent applicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which this invention pertains. Allpublications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. The use of the word “a” or “an” when used inconjunction with the term “comprising” in the claims and/or thespecification may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.” The use ofthe term “or” in the claims is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive, although the disclosure supports a definition that refers toonly alternatives and “and/or.” Throughout this application, the term“about” is used to indicate that a value includes the inherent variationof error for the device, the method being employed to determine thevalue, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps

Any part of this disclosure may be read in combination with any otherpart of the disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

The present invention is described in more detail in the following nonlimiting Examples.

The invention addresses the need to treat humans havingnaturally-occurring rarer natural PCSK9 alleles, genotypes andphenotypes (rarer protein forms). In this respect, the inventionprovides the following aspects.

In a First Aspect: A method of reducing cholesterol level or maintainingpreviously reduced cholesterol level in a human in need thereofcomprising a. selecting a human comprising (i) a proprotein convertasesubtilisin/kexin type 9 (PCSK9) nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 29-37; and/or (ii) a nucleotide sequencethereof encoding the catalytic domain or C-terminal domain of a PCSK9protein; and b. administering to said human an antibody or antibodyfragment that specifically binds one or more a PCSK9 amino acid sequenceencoded by said nucleotide sequence comprised by the human.

In an example, step (a) comprises selecting a human comprising a PCSK9protein encoded by the nucleotide sequence of (i) or (ii).

In an example, the antibody or antibody fragment specifically binds saidPCSK9 amino acid sequence. In an example, the antibody or antibodyfragment binds a second PCSK9 protein comprising an amino acid sequenceencoded by (i) a nucleotide sequence selected from the group consistingof SEQ ID NOs: 29-37; and/or (ii) a nucleotide sequence thereof encodingthe catalytic domain or C-terminal domain of a PCSK9 protein. In anexample, the antibody comprises a VH domain derived from therecombination of a human VH gene segment, human D gene segments and ahuman JH segment, the VH gene segment comprising a nucleotide sequencethat comprises a single nucleotide polymorphism with nucleotide C asindicated in rs56069819(CTGTACCAAGCCTCCCCCAGACTCCA[A/C]CAGCTGCACCTCACACTGGACACCT (SEQ ID NO:41)). In an example, the VH gene segment is VH3-23*04 (SEQ ID NO: 38)encoded by a sequence comprising SEQ ID NO: 39. In an example, theantibody comprises a VH domain, wherein the VH domain comprises theframework 1 sequence of SEQ ID No. 40.

In an example, the human has been determined to comprise the nucleotidesequence of (i) and/or (ii). In an example, the human has beendetermined to comprise a proprotein convertase subtilisin/kexin type 9(PCSK9) variant protein encoded by the nucleotide sequence of (i) and/or(ii). In an example, the method further comprises the step ofdetermining that the human comprises the nucleotide sequence of (i)and/or (ii).

In an example, the determining step is performed before administrationof the antibody to the human. In an example, the method furthercomprises the step of determining that the human comprises a proproteinconvertase subtilisin/kexin type 9 (PCSK9) variant protein encoded bythe nucleotide sequence of (i) and/or (ii). In an example, thedetermining step is performed before administration of the antibody tothe human. In an example, the step of determining comprises assaying abiological sample from the human for (i) a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 29-37; and/or (ii) a nucleotidesequence encoding the catalytic domain or C-terminal domain of the PCSK9variant protein. In an example, the assaying comprises contacting thebiological sample with c. at least one oligonucleotide probe comprisinga sequence of at least 10 contiguous nucleotides that can specificallyhybridize to and identify in the biological sample a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 29-37 or at least thecatalytic domain- or C-terminal domain-encoding sequence thereof, orthat specifically hybridizes to an antisense of said sequence, whereinsaid nucleic acid hybridizes to at least one nucleotide present in saidselected sequence which is not present in SEQ ID NO: 28 or hybridizes toan antisense sequence thereby forming a complex when at least onenucleotide sequence selected from the group consisting of SEQ ID NOs:29-37 or at least the catalytic domain- or C-terminal domain-encodingsequence thereof is present; and/or d. at least one oligonucleotideprobe comprising a sequence of at least 10 contiguous nucleotides of anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37 or comprising an antisense sequence of said contiguousnucleotides, wherein said sequence of contiguous nucleotides comprisesat least one nucleotide present in said selected sequence which is notpresent in SEQ ID NO: 28 thereby forming a complex when a nucleotidesequence selected from the group consisting of SEQ ID NOs: 29-37 ispresent; and detecting the presence or absence of the complex, whereindetecting the presence of the complex determines that the humancomprises the PCSK9 variant protein.

In an example, the assaying comprises nucleic acid amplification andoptionally one or more methods selected from sequencing, next generationsequencing, nucleic acid hybridization, and allele-specificamplification. In an example, the assaying is performed in a multiplexformat. In an example the method further comprises obtaining thebiological sample from the human. In an example, said biological samplecomprises serum, blood, feces, tissue, a cell, urine and/or saliva ofsaid human.

In an example, said human is or has been further determined to besubstantially resistant to statin treatment. In an example, the human isreceiving or has received statin treatment or has reduced responsivenessto statin treatment.

In an example, the human is further administered a statin. In anexample, said antibody or antibody fragment and said statin areadministered separately or simultaneously.

In an example, said human is indicated as heterozygous for a nucleotidesequence selected from the group consisting of SEQ ID NOs: 29-37 and/orthe nucleotide sequence thereof encoding the catalytic domain- orC-terminal domain-encoding sequence of a PCSK9 protein. In an example,said human is further indicated as comprising the nucleotide sequence ofSEQ ID NO: 28 and/or the catalytic domain- or C-terminal domain-encodingsequence thereof.

In an example, said human is indicated as homozygous for a nucleotidesequence selected from the group consisting of SEQ ID NOs: 29-37 and/orthe catalytic domain- or C-terminal domain-encoding sequence thereof.

In an example, said human has been diagnosed with at least one conditionselected from a lipid disorder, hyperlipoproteinemia, hyperlipidemia;dyslipidemia; hypercholesterolemia, a heart attack, a stroke, coronaryheart disease, atherosclerosis, peripheral vascular disease,claudication, type II diabetes, high blood pressure, and acardiovascular disease or condition.

In an example, said method treats or prevents in said human at least onecondition selected from a lipid disorder, hyperlipoproteinemia,hyperlipidemia; dyslipidemia; hypercholesterolemia, a heart attack, astroke, coronary heart disease, atherosclerosis, peripheral vasculardisease, claudication, type II diabetes, high blood pressure, and acardiovascular disease or condition.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. A method of treating and/or preventing a disease or condition        mediated by proprotein convertase subtilisin/kexin type 9        (PCSK9) in a human who has been determined to comprise and/or        selected as comprising a PCSK9 variant protein, the method        comprising administering to the human a ligand that binds the        PCSK9 variant protein to treat and/or prevent said disease or        condition.    -   2. The method of paragraph 1, wherein said PCSK9 variant protein        is selected from the group consisting of PCSK9 variant protein        forms f, c, r, p, m, e, h, aj and q.    -   3. The method of paragraph 2, wherein the variant is mature        PCSK9.    -   4. The method of paragraph 1, 2 or 3 further comprising assaying        a biological sample from the human for the PCSK9 variant protein        form.    -   5. A method of treating and/or preventing a disease or condition        mediated by proprotein convertase subtilisin/kexin type 9        (PCSK9) variant protein encoded by (i) a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 29-37;        and/or (ii) a nucleotide sequence encoding the catalytic domain        or C-terminal domain thereof in a human who has been determined        as comprising and/or selected as comprising (i) the nucleotide        sequence selected from the group consisting of SEQ ID NOs:        29-37; and/or (ii) the nucleotide sequence thereof encoding the        catalytic domain or C-terminal domain of a PCSK9 protein, the        method comprising administering to said human a ligand that        binds said PCSK9 variant protein to treat and/or prevent said        disease or condition.    -   6. The method of paragraph 5 further comprising assaying a        biological sample from the human for (i) a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 29-37;        and/or (ii) a nucleotide sequence thereof encoding the catalytic        domain or C-terminal domain of a PCSK9 protein.    -   7. The method of paragraph 6, wherein the assaying comprises        nucleic acid amplification and/or one or more methods selected        from sequencing, next generation sequencing, nucleic acid        hybridization, and allele-specific amplification.    -   8. The method of paragraph 6, wherein the assaying is performed        in a multiplex format.    -   9. The method of any one of paragraphs 1-8, further comprising        obtaining the biological sample from the human.    -   10. The method of any one of paragraphs 1-9, wherein said human        has been further determined to be and/or selected as        substantially resistant to statin treatment of said disease or        condition.    -   11. The method of any one of paragraphs 1-10, wherein the ligand        is selected from an antibody, an antibody portion, an antibody        fragment or an affibody.    -   12. The method of paragraph 11, wherein the ligand is an        antibody or antibody fragment that specifically binds to a human        PCSK9 selected from forms f, c, m, e, h, p, q and aj, wherein        the antibody or fragment comprises a VH domain derived from the        recombination of a human VH gene segment, a human D gene segment        and a human JH gene segment, the VH gene segment comprising a        nucleotide sequence that comprises SNP rs56069819 (SEQ ID NO:        41).    -   13. The ligand of paragraph 12, wherein the VH gene segment is        VH3-23*04.    -   14. The method of any one of the paragraphs 1-13, wherein said        administering further comprises administering a statin to the        human.    -   15. The method of any one of paragraphs 1-14, wherein said        ligand and said statin are administered separately or        simultaneously.    -   16. The method of any one of paragraphs 1-15, wherein said        biological sample comprises serum, blood, feces, hair, tissue,        cells, urine and/or saliva of said human.    -   17. The method of any one of the paragraphs 1-16, wherein said        human is indicated as heterozygous for a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 29-37 and/or        the nucleotide sequence thereof encoring a catalytic domain- or        C-terminal domain-encoding sequence of a PCSK9 protein.    -   18. The method of any one of paragraphs 1-17, wherein said human        is indicated as comprising the nucleotide sequence of SEQ ID NO:        28 and/or the catalytic domain- or C-terminal domain-encoding        sequence thereof.    -   19. The method of any one of paragraphs 1-18, wherein said human        is indicated as homozygous for a nucleotide sequence selected        from the group consisting of SEQ ID NOs: 29-37 and/or the        nucleotide sequence thereof encoding a catalytic domain- or        C-terminal domain-encoding sequence of a PCSK2 protein.    -   20. The method of any one of paragraphs 1-19, wherein when said        human is determined to comprise and/or selected as comprising:        -   a. SEQ ID NO: 29 and is classified as of ASW, YRI, GBR, TSI,            CLM, LWK, MXL, JPT, PUR, IBS, FIN or CEU ancestry, then            administering to said human a ligand that specifically binds            said PCSK9 variant protein comprising a variant encoded by            SEQ ID NO: 29; or        -   b. SEQ ID NO: 30 and is classified as of ASW, YRI, GBR, TSI,            CLM, CHB, LWK, CHS, JPT, PUR, FIN or CEU ancestry then            administering to said human a ligand that specifically binds            said PCSK9 variant protein comprising a variant encoded by            SEQ ID NO: 30; or        -   c. SEQ ID NO: 32, and is classified as of ASW, GBR, TSI,            CLM, JPT, PUR, IBS, FIN or CEU ancestry then administering            to said human a ligand that specifically binds said PCSK9            variant protein comprising a variant encoded by SEQ ID NO:            32; or        -   d. SEQ ID NO: 33, and is classified as of LWK, ASW, YRI or            CLM ancestry then administering to said human a ligand that            specifically binds said PCSK9 variant protein comprising a            variant encoded by SEQ ID NO: 33; or        -   e. SEQ ID NO: 34, and is classified as of LWK, ASW or YRI            ancestry then administering to said human a ligand that            specifically binds said PCSK9 variant protein comprising a            variant encoded by SEQ ID NO: 34; or        -   f. SEQ ID NO: 35, and is classified as of PUR, TSI, FIN or            CEU ancestry then administering to said human a ligand that            specifically binds said PCSK9 variant protein comprising a            variant encoded by SEQ ID NO: 35; or        -   g. SEQ ID NO: 36, and is classified as of LWK, ASW or YRI            ancestry then administering to said human a ligand that            specifically binds said PCSK9 variant protein comprising a            variant encoded by SEQ ID NO: 36; or        -   h. SEQ ID NO: 37, and is classified as of CHS, ASW, JPT, PUR            or CHB ancestry then administering to said human a ligand            that specifically binds said PCSK9 variant protein            comprising a variant encoded by SEQ ID NO: 37.    -   21. The method of any one of paragraphs 1-19, wherein when said        human is determined to comprise and/or selected as comprising:        -   a. PCSK9 protein form f, and is classified as of ASW, YRI,            GBR, TSI, CLM, LWK, MXL, JPT, PUR, IBS, FIN or CEU ancestry            then administering a ligand that specifically binds said            PCSK9 protein form f for a time and in an amount effective            to treat and/or prevent said disease or condition in said            human, thereby to treat and/or prevent said disease or            condition in said human; or        -   b. PCSK9 protein form c, and is classified as of ASW, YRI,            GBR, TSI, CLM, CHB, LWK, CHS, JPT, PUR, FIN or CEU ancestry            then administering a ligand that specifically binds said            PCSK9 protein form c for a time and in an amount effective            to treat and/or prevent said disease or condition in said            human, thereby to treat and/or prevent said disease or            condition in said human; or        -   c. PCSK9 protein form p, and is classified as of ASW, GBR,            TSI, CLM, JPT, PUR, IBS, FIN or CEU ancestry then            administering a ligand that specifically binds said PCSK9            protein form p for a time and in an amount effective to            treat and/or prevent said disease or condition in said            human, thereby to treat and/or prevent said disease or            condition in said human; or        -   d. PCSK9 protein form m, and is classified as of LWK, ASW,            YRI or CLM ancestry then administering a ligand that            specifically binds said PCSK9 protein form m for a time and            in an amount effective to treat and/or prevent said disease            or condition in said human, thereby to treat and/or prevent            said disease or condition in said human; or        -   e. PCSK9 protein form e, and is classified as of LWK, ASW or            YRI ancestry then administering a ligand that specifically            binds said PCSK9 protein form e for a time and in an amount            effective to treat and/or prevent said disease or condition            in said human, thereby to treat and/or prevent said disease            or condition in said human; or        -   f. PCSK9 protein form h, and is classified as of PUR, TSI,            FIN or CEU ancestry then administering a ligand that            specifically binds said PCSK9 protein form h for a time and            in an amount effective to treat and/or prevent said disease            or condition in said human, thereby to treat and/or prevent            said disease or condition in said human; or        -   g. PCSK9 protein form aj, and is classified as of LWK, ASW            or YRI ancestry then administering a ligand that            specifically binds said PCSK9 protein form aj for a time and            in an amount effective to treat and/or prevent said disease            or condition in said human, thereby to treat and/or prevent            said disease or condition in said human; or        -   h. PCSK9 protein form q, and is classified as of CHS, ASW,            JPT, PUR or CHB ancestry then administering a ligand that            specifically binds said PCSK9 protein form q for a time and            in an amount effective to treat and/or prevent said disease            or condition in said human, thereby to treat and/or prevent            said disease or condition in said human.    -   22. The method of any one of paragraphs 1-21, wherein said        ligand is capable of specifically binding said PCSK9 variant        protein or a nucleic acid encoding said PCSK9 variant protein.    -   23. The method of any one of paragraphs 1-21, wherein said        ligand specifically binds two or more human PCSK9 variant        proteins or fragment thereof selected from the group consisting        of SEQ ID NOs: 4-27.    -   24. The method of paragraph 23, wherein said ligand specifically        binds two or more human PCSK9 proteins or fragment thereof,        wherein at least one of the protein fragments comprise an amino        acid sequence selected from SEQ ID NOs: 4-14, 18-23, 26 and 27.    -   25. The method of any one of paragraphs 4 and 6-23, wherein said        human PCSK9 protein assayed in said sample is in the mature        form.    -   26. The method of any one of paragraphs 4 and 6-24, wherein said        human PCSK9 protein assayed in said sample is in the pro-form.    -   27. The method of paragraph 1-26, wherein said disease or        condition is selected from a lipid disorder,        hyperlipoproteinemia, hyperlipidemia; dyslipidemia;        hypercholesterolemia, a heart attack, a stroke, coronary heart        disease, atherosclerosis, peripheral vascular disease,        claudication, type II diabetes, high blood pressure, and a        cardiovascular disease or condition.    -   28. A kit for genotyping a proprotein convertase        subtilisin/kexin type 9 (PCSK9) gene variant in a nucleic acid        sample of a human being affected with or at risk of a PCSK9        mediated disease, the kit comprising        -   a. at least one nucleic acid probe comprising a sequence of            at least 10 contiguous nucleotides that can specifically            hybridize to and identify in a biological sample the            presence of a nucleotide sequence selected from the group            consisting of SEQ ID NOs: 29-37 and the sequence thereof            encoding a catalytic domain- or C-terminal domain of a PCSK9            protein, or can specifically hybridize to and identify in a            biological sample the presence of an antisense of said            nucleotide sequence, wherein said nucleic acid probe            hybridizes to at least one nucleotide present in said            selected sequence which is not present in SEQ ID NO: 28 or            an antisense sequence thereof, and/or        -   b. at least two nucleic acid probes comprising at least two            sequences, each of at least 10 contiguous nucleotides of a            nucleotide sequence selected from the group consisting of            SEQ ID NOs: 29-37 or comprising an antisense sequence of            said contiguous nucleotides, wherein at least one of said            oligonucleotide probes comprises at least one nucleotide            present in said selected nucleotide sequence which is not            present in SEQ ID NO: 28 or an antisense sequence thereof,            and        -   c. optionally at least one of the following: one or more            buffers, packaging, label, and/or instructions for PCSK9            genotyping in a biological sample comprising nucleic acids            obtained from a human.    -   29. The kit of paragraph 28, wherein the nucleic acid probe is        attached to a solid surface.    -   30. The kit of any of paragraphs 28-29, further comprising a        detectable label attached to said nucleic acid probe.    -   31. A kit for phenotyping proprotein convertase subtilisin/kexin        type 9 (PCSK9) protein in a biological sample, the kit        comprising a plurality of antibodies or antibody portions or        fragments, wherein each antibody or antibody fragment or portion        is specific for a PCSK9 variant protein selected from the group        consisting of forms f, c, r, p, m, e, h, aj and q or a catalytic        or C-terminal domain or a peptide thereof that comprises amino        acid variation from the corresponding sequence of SEQ ID NO: 1,        2 or 3; and optionally at least one of the following: one or        more buffers, packaging and/or label or instructions or a label        for PCSK9 phenotyping in a biological sample comprising PCSK9        obtained from a human.    -   32. The kit of paragraph 31, further comprising a statin.    -   33. A pharmaceutical composition comprising a ligand capable of        specifically binding proprotein convertase subtilisin/kexin type        9 (PCSK9) variants selected from the group consisting of forms        f, c, r, p, m, e, h, aj and q or a catalytic or C-terminal        domain or a peptide thereof that comprises amino acid variation        from the corresponding sequence of SEQ ID NO: 1, 2 or 3.    -   34. The pharmaceutical composition of paragraph 33, wherein the        ligand is an antibody, antibody portion, antibody fragment or an        affibody specific for the PCSK9 variant selected from the group        consisting of forms f, c, r, p, m, e, h, aj and q or a catalytic        or C-terminal domain or a peptide thereof that comprises amino        acid variation from the corresponding sequence of SEQ ID NO: 1,        2 or 3.    -   35. The pharmaceutical composition of any of paragraphs 33-34,        further comprising a statin.    -   36. The pharmaceutical composition of paragraph 35, wherein the        statin is atorvastatin.    -   37. A drug delivery system comprising the pharmaceutical        composition of paragraph 33 or 34 and an injection or IV device.    -   38. A kit comprising the pharmaceutical composition of paragraph        33, packaging and instructions or label for administering the        ligand to a human affected with or at risk of a PCSK9-mediated        disease or condition, wherein the human expresses a variant        PCSK9 protein form selected from the group consisting of forms        f, c, r, p, m, e, h, aj and q or a catalytic or C-terminal        domain or a peptide thereof.    -   39. The kit of paragraph 38, wherein the instructions or label        indicate administration with a statin and/or to a human who is        indicated for statin treatment or has been administered a statin        or has reduced responsiveness to statin treatment.    -   40. The kit of paragraph 38 or 39, wherein the human has been        determined to be and/or selected as resistant to treatment with        statins.    -   41. The kit of paragraph 39 or 40, wherein the instructions or        label indicate administration to a human who has received statin        and further instruct reducing statin administration to the        human.    -   42. The kit of paragraph 38, further comprising at least one        buffer, packaging and/or instructions or label for PCSK9        genotyping in a biological sample obtained from a human.    -   43. The kit of paragraph 38 wherein the label comprises a        marketing authorization number issued by a regulatory agency.    -   44. The kit of paragraph 43, wherein the regulatory agency is        selected from the FDA or EMA.    -   45. The kit of paragraph 38, wherein the label or instructions        further comprise directions to administer alirocumab or        evolocumab to said human.    -   46. The kit of any one of paragraphs 38-45, further comprising        an IV or injection device that optionally comprises alirocumab        or evolocumab.    -   47. A kit for treating and/or preventing a disease or condition        mediated by proprotein convertase subtilisin/kexin type 9        (PCSK9) in a human who has been determined to comprise and/or        selected as comprising a PCSK9 variant protein the kit        comprising (a) a alirocumab or evolocumab in a sealed container,        and (b) a label or instructions indicating administration of the        alirocumab or evolocumab to a human who expresses a variant        PCSK9 protein form selected from the group consisting of forms        f, c, r, p, m, e, h, aj and q or a catalytic or C-terminal        domain or a peptide thereof.    -   48. The kit of paragraph 47, wherein the label further indicates        that if the human has been or is administered statin, the statin        treatment should be continued.    -   49. The kit of paragraph 47, wherein the label further indicates        that if the human has been or is administered statin, the statin        treatment should be reduced or discontinued.    -   50. A method of treating and/or preventing a disease or        condition mediated by proprotein convertase subtilisin/kexin        type 9 (PCSK9) in a human, the method comprising        -   a. assaying a biological sample from the human for the            presence of one or more PCSK9 protein variants selected from            the group consisting of PCSK9 forms f, c, r, p, m, e, h, aj            and q; and        -   b. administering a therapeutically effective amount of a            human PCSK9 binding ligand to the human when one or more of            the PCSK9 forms f, c, r, p, m, e, h, aj and q is detected.    -   51. The method of paragraph 50, wherein the ligand specifically        binds one or more of the PCSK9 forms f, c, r, p, m, e, h, aj and        q.    -   52. A method of treating and/or preventing a disease or        condition mediated by proprotein convertase subtilisin/kexin        type 9 (PCSK9) in a human, the method comprising        -   a. assaying a biological sample from the human for the            presence or one or more PSCK9 nucleic acid variants selected            from the group consisting of SEQ ID NOs: 29-37 or at least            the catalytic domain- or C-terminal domain-encoding sequence            thereof, and        -   b. administering a therapeutically effective amount of a            human PCSK9 binding ligand to the human when one or more of            the PCSK9 nucleic acid variants are detected.    -   53. The method of paragraph 52, wherein the ligand specifically        binds one or more of the PCSK9 forms f, c, r, p, m, e, h, aj and        q.    -   54. A method of selecting a human for treatment and/or        prevention of a disease mediated by a proprotein convertase        subtilisin/kexin type 9 (PCSK9) protein with a human PCSK9        binding ligand comprising:        -   a. assaying a biological sample taken from the human for the            presence of a PCSK9 protein variant selected from the group            consisting of forms f, c, r, p, m, e, h, aj and q, and        -   b. selecting the human for treatment with the human PCSK9            binding ligand when at least one of the PCSK9 protein            variants selected from the group consisting of forms f, c,            r, p, m, e, h, aj and q is detected.    -   55. The method of paragraph 54, wherein the human is indicated        for statin treatment or has been administered statin.    -   56. The method of paragraph 54 or 55, wherein the human has been        identified as substantially resistant to statin treatment or has        reduced responsiveness to statin treatment.    -   57. The method of any one of paragraphs 54-56, wherein the        disease or condition is selected from a lipid disorder,        hyperlipoproteinemia, hyperlipidemia; dyslipidemia;        hypercholesterolemia, heart attack, stroke, coronary heart        disease, atherosclerosis, peripheral vascular disease,        claudication, type II diabetes, high blood pressure, and a        cardiovascular disease or condition.    -   58. The method of any one of paragraphs 54-57, wherein the        administered human PCSK9 binding ligand is specific for the        detected PCSK9 form.    -   59. The method of any one of paragraphs 54-58, wherein said        PCSK9 protein form comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs: 4-27.    -   60. The method of any one of paragraphs 54-59, wherein said        PCSK9 protein form comprises the mature PCSK9 form.    -   61. An assay for selecting a human affected with a proprotein        convertase subtilisin/kexin type 9 (PCSK9) protein mediated        disease or condition as eligible for treatment with human PCSK9        binding ligand, the method comprising        -   a. contacting a biological sample from said human with at            least one oligonucleotide probe comprising a sequence of at            least 10 contiguous nucleotides that can specifically            hybridize to and identify in the biological sample a            nucleotide sequence selected from the group consisting of            SEQ ID NOs: 29-37 or at least the catalytic domain- or            C-terminal domain-encoding sequence thereof, or that            specifically hybridizes to an antisense of said sequence,            wherein said nucleic acid hybridizes to at least one            nucleotide present in said selected sequence which is not            present in SEQ ID NO: 28 or hybridizes to an antisense            sequence thereby forming a complex when at least one            nucleotide sequence selected from the group consisting of            SEQ ID NOs: 29-37 or at least the catalytic domain- or            C-terminal domain-encoding sequence thereof is present;            and/or        -   b. detecting the presence or absence of the complex; and        -   c. selecting the human as eligible for treatment with the            human PCSK9 binding ligand when presence of at least one            complex comprising a PCSK9 form encoded by SEQ ID NOs: 29-37            or the catalytic domain- or C-terminal domain-encoding            sequence thereof is detected.    -   62. An assay for selecting a human as eligible for treatment        with human proprotein convertase subtilisin/kexin type 9 (PCSK9)        binding ligand, the method comprising        -   a. contacting a biological sample from a human with PCSK9            mediated disease or condition with one or more antibody,            antibody portion or antibody fragment capable of binding to            a PCSK9 variant form selected from the group consisting of            PCSK9 form f, c, r, p, m, e, h, aj and q thereby forming a            complex when one or more PCSK9 form f, c, r, p, m, e, h, aj            and q is present;        -   b. detecting the presence or absence or the complex; and        -   c. selecting the human as eligible for treatment with the            human PCSK9 binding ligand when presence of at least one            complex comprising PCSK9 form f, c, r, p, m, e, h, aj and q            is detected.    -   63. The assay of paragraphs 61 or 62, wherein the disease or        condition is selected from a lipid disorder,        hyperlipoproteinemia, hyperlipidemia; dyslipidemia;        hypercholesterolemia, heart attack, stroke, coronary heart        disease, atherosclerosis, peripheral vascular disease,        claudication, type II diabetes, high blood pressure, and a        cardiovascular disease or condition.    -   64. The assay of any one of paragraphs 61-63, wherein the human        PSCK9 binding ligand is specific for the detected PCSK9 form.    -   65. The assay of any one of paragraphs 61-64, further comprising        amplifying nucleic acids from the biological sample.    -   66. The assay of any one of paragraphs 61-65, further comprising        isolating nucleic acids from the biological sample.    -   67. The assay of any one of paragraphs 61-66, further comprising        administering to said human the PCSK9-binding ligand.    -   68. A method of producing an anti-human proprotein convertase        subtilisin/kexin type 9 (PCSK9) antibody binding site, the        method comprising obtaining a plurality of anti-PCSK9 antibody        binding sites, screening the antibody binding sites for binding        to a human PCSK9 selected from the group consisting of forms f,        c, r, p, m, e, h, aj and q or a catalytic or C-terminal domain        or a peptide thereof that comprises amino acid variation from        the corresponding sequence of SEQ ID NO: 1, 2 or 3 and isolating        an antibody binding site that binds in the screening step, and        optionally producing a form f, c, r, p, m, e, h, aj or q        PCSK9-binding fragment or derivative of the isolated antibody.    -   69. A method of producing an anti-human proprotein convertase        subtilisin/kexin type 9 (PCSK9) antibody, the method comprising        immunising a non-human vertebrate with a human PCSK9 comprising        an amino acid sequence selected from the group consisting of the        amino acid sequences of forms f, c, r, p, m, e, h, aj and q or a        catalytic or C-terminal domain or a peptide thereof that        comprises amino acid variation from the corresponding sequence        of SEQ ID NO: 1, 2 or 3 and isolating an antibody that binds a        human PCSK9 comprising selected from the group consisting of        forms f, c, r, p, m, e, h, aj and q or a catalytic or C-terminal        domain or a peptide thereof that comprises amino acid variation        from the corresponding sequence of SEQ ID NO: 1, 2 or 3, and        optionally producing a form f, c, r, p, m, e, h, aj or q        PCSK9-binding fragment or derivative of the isolated antibody.    -   70. The method of paragraphs 68-69, wherein the non-human        vertebrate is a mouse or a rat.    -   71. The method of any of paragraphs 68-70, comprising the step        of obtaining a nucleic acid encoding the antibody, fragment,        derivative or binding site and optionally inserting the nucleic        acid in an expression vector.    -   72. A kit for proprotein convertase subtilisin/kexin type 9        (PCSK9) genotyping a human, wherein the kit comprises a nucleic        acid (i) comprising a sequence of contiguous nucleotides that        specifically hybridises to a nucleotide sequence selected from        the group consisting of SEQ ID NOs: 29-37 or at least the        catalytic domain- or C-terminal domain-encoding sequence        thereof, or specifically hybridises to an antisense sequence or        an RNA transcript of said sequence, wherein said sequence of        contiguous nucleotides hybridises to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridises to an antisense sequence or an RNA        transcript thereof, and/or (ii) comprising a sequence of at        least 10 contiguous nucleotides of a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 29-37 or        comprising an antisense sequence or RNA version of said        contiguous nucleotides, wherein said sequence of contiguous        nucleotides comprises at least one nucleotide present in said        selected sequence which is not present in SEQ ID NO: 28.    -   73. A kit for proprotein convertase subtilisin/kexin type 9        (PCSK9) genotyping or phenotyping a human, wherein the kit        comprises a ligand antibody that binds a human PCSK9 comprising        selected from the group consisting of forms f, c, r, p, m, e, h,        aj and q or a catalytic or C-terminal domain or a peptide        thereof that comprises amino acid variation from the        corresponding sequence of SEQ ID NO: 1, 2 or 3, or an antibody,        fragment or derivative produced by the method of any one of        paragraphs 68-71.    -   74. A method of targeting a proprotein convertase        subtilisin/kexin type 9 (PCSK9) for treating and/or preventing a        PCSK9-mediated disease or condition in a human, the method        comprising administering an anti-PCSK9 ligand to a human        comprising a nucleotide sequence selected from the group        consisting SEQ ID NOs: 29-37, whereby a PCSK9 encoded by said        nucleotide sequence is targeted.    -   75. The method of paragraph 74, wherein the method comprises        targeting a human PCSK9 selected from the group consisting of        forms f, c, r, p, m, e, h, aj and q with said ligand to treat        and/or prevent said disease or condition in said human.    -   76. A method of treating and/or preventing a disease or        condition mediated by proprotein convertase subtilisin/kexin        type 9 (PCSK9) in a human, the method comprising targeting a        human PCSK9 selected from the group consisting of forms f, c, r,        p, m, e, h, aj and q by administering to the human a ligand that        binds said PCSK9 thereby treating and/or preventing said disease        or condition in the human.    -   77. The method of paragraph 76, wherein the genome of the human        comprises a nucleotide sequence selected from the group        consisting of SEQ ID NOs: 29-37.    -   78. The method of any one of paragraphs 74 to 77, wherein the        human has been or is genotyped as positive for a nucleotide        sequence selected from the group consisting of SEQ ID NOs: 29-37        or the catalytic- or C-terminal domain-encoding sequence        thereof.    -   79. The method of any one of paragraphs 74 to 78, wherein the        human has been or is phenotyped as positive for a human PCSK9        selected from the group consisting of forms f, c, r, p, m, e, h,        aj and q.    -   80. The method of any one of paragraphs 74-79, wherein the        method comprises genotyping the human as positive for a        nucleotide sequence selected from the group consisting of SEQ ID        NOs: 29-37 or the catalytic- or C-terminal domain-encoding        sequence thereof.    -   81. The method of any one of paragraphs 74-80, wherein the        method comprises phenotyping the human as positive for a human        PCSK9 sequence selected from the group consisting of forms f, c,        r, p, m, e, h, aj and q.    -   82. The method of any one of paragraphs 74 to 81, wherein the        human has been or is genotyped as heterozygous for a nucleotide        sequence selected from the group consisting of SEQ ID NOs: 29-37        or the catalytic- or C-terminal domain-encoding sequence        thereof; optionally wherein the human has been or is genotyped        as comprising the nucleotide sequence of SEQ ID NO: 28 or the        catalytic- or C-terminal domain-encoding sequence thereof and a        nucleotide sequence selected from the group consisting of SEQ ID        NOs: 29-37 or the catalytic- or C-terminal domain-encoding        sequence thereof.    -   83. The method of any one of paragraphs 74 to 82, wherein the        genome of the human has been or is genotyped as homozygous for a        nucleotide sequence selected from the group consisting of SEQ ID        NOs: 29-37 or the catalytic- or C-terminal domain-encoding        sequence thereof.    -   84. The method of any one of paragraphs 74 to 83, wherein the        method comprises genotyping the human for a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 29-37 or the        catalytic- or C-terminal domain-encoding sequence thereof before        administering the ligand to the human, wherein the ligand is        determined to be capable of binding to a PCSK9 encoded by said        selected sequence.    -   85. The method according to any one of paragraphs 74 to 84        further comprising administering said ligand and a statin (eg,        atorvastatin) to the human.    -   86. The method of paragraph 85, wherein the ligand and statin        are administered separately.    -   87. The method of paragraph 85, wherein the ligand and statin        are administered simultaneously.    -   88. The method of any one of paragraphs 74 to 87, wherein the        ligand is administered by subcutaneous injection.    -   89. A method of reducing cholesterol level or maintaining        previously reduced cholesterol level in a human in need thereof        comprising        -   a. selecting a human comprising (i) a proprotein convertase            subtilisin/kexin type 9 (PCSK9) nucleotide sequence selected            from the group consisting of SEQ ID NOs: 29-37; and/or (ii)            a nucleotide sequence thereof encoding the catalytic domain            or C-terminal domain of a PCSK9 protein; and        -   b. administering to said human an antibody or antibody            fragment that specifically binds one or more a PCSK9 amino            acid sequence encoded by said nucleotide sequence comprised            by the human.    -   90. The method of paragraph 89, wherein step (a) comprises        selecting a human comprising a PCSK9 protein encoded by the        nucleotide sequence of (i) or (ii).    -   91. The method of paragraph 89 or 90, wherein the antibody or        antibody fragment specifically binds said PCSK9 amino acid        sequence.    -   92. The method of paragraph 89 or 90, wherein the antibody or        antibody fragment binds a second PCSK9 protein comprising an        amino acid sequence encoded by (i) a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 29-37;        and/or (ii) a nucleotide sequence thereof encoding the catalytic        domain or C-terminal domain of a PCSK9 protein.    -   93. The method of paragraph 89 or 90, wherein the antibody        comprises a VH domain derived from the recombination of a human        VH gene segment, human D gene segments and a human JH segment,        the VH gene segment comprising a nucleotide sequence that        comprises a single nucleotide polymorphism with nucleotide C as        indicated in rs56069819 (SEQ ID NO 41).    -   94. The method of paragraph 93, wherein the VH gene segment is        VH3-23*04.    -   95. The method of any one of paragraphs 89-93, wherein the        antibody comprises a VH domain, wherein the VH domain comprises        the framework 1 sequence of SEQ ID NO: 38.    -   96. The method of any one of paragraphs 89-95, wherein the human        has been determined to comprise the nucleotide sequence of (i)        and/or (ii).    -   97. The method of any one of paragraphs 89-96, wherein the human        has been determined to comprise a proprotein convertase        subtilisin/kexin type 9 (PCSK9) variant protein encoded by the        nucleotide sequence of (i) and/or (ii).

The method of any one of paragraphs 89-97, comprising the step ofdetermining that the human comprises the nucleotide sequence of (i)and/or (ii).

-   -   98. The method of paragraph 98, wherein the determining step is        performed before administration of the antibody to the human.    -   99. The method of any one of paragraphs 89-99, comprising the        step of determining that the human comprises a proprotein        convertase subtilisin/kexin type 9 (PCSK9) variant protein        encoded by the nucleotide sequence of (i) and/or (ii).    -   100. The method of paragraph 100, wherein the determining step        is performed before administration of the antibody to the human.    -   101. The method of paragraph 100 or 101, wherein the step of        determining comprises assaying a biological sample from the        human for (i) a nucleotide sequence selected from the group        consisting of SEQ ID NOs: 29-37; and/or (ii) a nucleotide        sequence encoding the catalytic domain or C-terminal domain of        the PCSK9 variant protein.    -   102. The method of paragraph 102, wherein the assaying comprises        contacting the biological sample with        -   a. at least one oligonucleotide probe comprising a sequence            of at least 10 contiguous nucleotides that can specifically            hybridize to and identify in the biological sample a            nucleotide sequence selected from the group consisting of            SEQ ID NOs: 29-37 or at least the catalytic domain- or            C-terminal domain-encoding sequence thereof, or that            specifically hybridizes to an antisense of said sequence,            wherein said nucleic acid hybridizes to at least one            nucleotide present in said selected sequence which is not            present in SEQ ID NO: 28 or hybridizes to an antisense            sequence thereby forming a complex when at least one            nucleotide sequence selected from the group consisting of            SEQ ID NOs: 29-37 or at least the catalytic domain- or            C-terminal domain-encoding sequence thereof is present;            and/or        -   b. at least one oligonucleotide probe comprising a sequence            of at least 10 contiguous nucleotides of a nucleotide            sequence selected from the group consisting of SEQ ID NOs:            29-37 or comprising an antisense sequence of said contiguous            nucleotides, wherein said sequence of contiguous nucleotides            comprises at least one nucleotide present in said selected            sequence which is not present in SEQ ID NO: 28 thereby            forming a complex when a nucleotide sequence selected from            the group consisting of SEQ ID NOs: 29-37 is present; and            detecting the presence or absence of the complex, wherein            detecting the presence of the complex determines that the            human comprises the PCSK9 variant protein.    -   103. The method of paragraph 102 or 103, wherein the assaying        comprises nucleic acid amplification and optionally one or more        methods selected from sequencing, next generation sequencing,        nucleic acid hybridization, and allele-specific amplification.    -   104. The method of any one of paragraphs 102-104, wherein the        assaying is performed in a multiplex format.    -   105. The method of any one of paragraphs 102-105, further        comprising obtaining the biological sample from the human.    -   106. The method of any one of paragraphs 89-106, wherein said        human is or has been further determined to be substantially        resistant to statin treatment.    -   107. The method of any one of paragraphs 89-107, wherein the        human is receiving or has received statin treatment or has        reduced responsiveness to statin treatment.    -   108. The method of any one of paragraphs 89-108, wherein the        human is further administered a statin.    -   109. The method of any one of paragraphs 89-108, wherein said        antibody or antibody fragment and said statin are administered        separately or simultaneously.    -   110. The method of any one of paragraphs 106-110, wherein said        biological sample comprises serum, blood, feces, tissue, a cell,        urine and/or saliva of said human.    -   111. The method of any one of paragraphs 89-111, wherein said        human is indicated as heterozygous for a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 29-37 and/or        the nucleotide sequence thereof encoding the catalytic domain-        or C-terminal domain-encoding sequence of a PCSK9 protein.    -   112. The method of any one of paragraphs 89-112, wherein said        human is further indicated as comprising the nucleotide sequence        of SEQ ID NO: 28 and/or the catalytic domain- or C-terminal        domain-encoding sequence thereof.    -   113. The method of any one of paragraphs 89-113, wherein said        human is indicated as homozygous for a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 29-37 and/or        the catalytic domain- or C-terminal domain-encoding sequence        thereof.    -   114. The method of any one of paragraphs 89-114, wherein said        human has been diagnosed with at least one condition selected        from a lipid disorder, hyperlipoproteinemia, hyperlipidemia;        dyslipidemia; hypercholesterolemia, a heart attack, a stroke,        coronary heart disease, atherosclerosis, peripheral vascular        disease, claudication, type II diabetes, high blood pressure,        and a cardiovascular disease or condition.    -   115. The method of any one of paragraphs 89-115, wherein said        method treats or prevents in said human at least one condition        selected from a lipid disorder, hyperlipoproteinemia,        hyperlipidemia; dyslipidemia; hypercholesterolemia, a heart        attack, a stroke, coronary heart disease, atherosclerosis,        peripheral vascular disease, claudication, type II diabetes,        high blood pressure, and a cardiovascular disease or condition.

EXAMPLES Example 1: Rare PCSK9 Variants

The present invention provides anti-PCSK9 ligands; and PCSK9-binding ortargeting ligands as described herein. The ligands have a variety ofutilities. Some of the ligands, for instance, are useful in specificbinding assays, for genotyping or phenotyping humans, affinitypurification of PCSK9, in particular human PCSK9 or its ligands and inscreening assays to identify other antagonists of PCSK9 activity. Someof the ligands of the invention are useful for inhibiting binding ofPCSK9 to LDLR, or inhibiting PCSK9-mediated activities.

Anti-PCSK9 ligands (eg, antibodies and anti-sense RNA) have beendeveloped based on targeting and neutralising so-called “wild-type”human PCSK9, which is a commonly-occurring form (see, eg,US20120093818A1 and US20110065902A1). While such therapies are usefulfor human patients harbouring this form of human PCSK9, the inventorconsidered it useful to investigate the possibility of targeting muchrarer—but still naturally-occurring—forms of PCSK9 amongst humanpopulations. In this way, the inventor arrived at insight into thenatural occurrences and distributions of rarer human PCSK9 forms thatcan serve as useful targets (at the protein or nucleic acid level) forhuman treatment, prophylaxis and diagnosis pertinent to diseases andconditions mediated or associated with PCSK9 activity. This particularlyprovides for tailored therapies, prophylaxis and diagnosis in humansthat are devoid of the common PCSK9 gene or protein (ie, the form a ora′ as used in US20120093818A1 and US20110065902A1 to generateantibodies).

The skilled person will know that SNPs or other changes that translateinto amino acid variation can cause variability in activity and/orconformation of human targets to be addressed. This has spawned greatinterest in personalized medicine where genotyping and knowledge ofprotein and nucleotide variability is used to more effectively tailormedicines and diagnosis of patients. The invention, therefore, providesfor tailored pharmaceuticals and testing that specifically addressesrarer PCSK9 polymorphic variant forms. Such forms or “alleles” (at thenucleotide level), in many of the examples determined by the inventor,comprise multiple changes at the nucleotide and amino acid levels fromthe corresponding common form nucleotide and amino acids sequences, ie,there are multiple non-synonymous changes at the nucleotide level thattranslate into multiple corresponding changes in the protein target inhumans.

Furthermore, the inventor surprisingly realised that the rarer naturalforms, although present in humans at much lower frequencies than thecommon form, nevertheless are represented in multiple andethnically-diverse human populations and usually with many humanexamples per represented ethnic population. Thus, the inventor realisedthat targeting such rarer forms would provide for effective treatment,prophylaxis or diagnosis across many human ethnic populations, therebyextending the utility of the present invention.

With this realisation, the inventor realised that there is significantindustrial and medical application for the invention in terms of guidingthe choice of anti-PCSK9 ligand for administration to human patients fortherapy and/or prophylaxis of PCSK9-mediated or associated diseases orconditions. In this way, the patient receives drugs and ligands that aretailored to their needs—as determined by the patient's genetic orphenotypic makeup. Hand-in-hand with this, the invention provides forthe genotyping and/or phenotyping of patients in connection with suchtreatment, thereby allowing a proper match of drug to patient. Thisincreases the chances of medical efficacy, reduces the likelihood ofinferior treatment using drugs or ligands that are not matched to thepatient (eg, poor efficacy and/or side-effects) and avoidspharmaceutical mis-prescription and waste.

In developing this thinking, in this non-limiting example the presentinventor decided to determine a set of human PCSK9 variants on the basisof the following criteria, these being criteria that the inventorrealised would provide for useful medical drugs and diagnostics totailored need in the human population. The inventor selected variantshaving at least 3 of the 4 following criteria:—

-   -   PCSK9 variants having a cumulative human allele frequency in the        range from 1 to 10%;    -   PCSK9 variants having a total human genotype frequency in the        range from 1 to about 15%;    -   PCSK9 variants found in many different human ethnic populations        (using the standard categorisation of the 1000 Genomes Project,        which is an accepted standard in the art; see Table 3 below);        and    -   PCSK9 variants found in many individuals distributed across such        many different ethnic populations.

On the basis of these criteria, the inventor identified the variantslisted in Table 1 below (excluding form a).

The inventor's selection included, as a consideration, selection fornucleotide variation that produced amino acid variation in correspondingPCSK9 forms (ie, non-synonymous variations), as opposed to silentvariations that do not alter amino acid residues in the target protein.

TABLE 1 Human PCSK9 variants distributed over several human ethnicpopulations & having a total human genotype frequency in the range of 1to about 15% (a) Amino acid variability, population distributions andfrequencies Form a 46R 53A 425N 443A 474I 619Q 670E ASW, YRI, GBR, 93914 0.3951 0.4506 0.64815 TSI, CLM, CHB, (0.8457) LWK, CHS, MXL, JPT,PUR, IBS, FIN, CEU No. Hom Freq⁴ Variant Amino Acid Position & VariationHuman No. Unique Het (Het + Hom Cum Form 46L 53V 425S 443T 474V 619P670G Populations Individs¹ Pops² Freq³ freq⁵) Freq⁶ f x ASW, YRI, GBR,180 12 0.153 0.009  0.0855 TSI, CLM, LWK, (0.162)  MXL, JPT, PUR, IBS,FIN, CEU c x ASW, YRI, GBR, 153 12 0.1296 0.0081 0.0729 TSI, CLM, CHB,(0.1377) LWK, CHS, JPT, PUR, FIN, CEU r x x 0.0234 0.009  0.0292(0.0324) p x x ASW, GBR, TSI, 49 9 0.0441 (0.0441) 0.0221 CLM, JPT, PUR,IBS, FIN, CEU m x LWK, ASW, YRI, 29 4 0.0225 (0.0225) 0.0149 CLM e x xLWK, ASW, YRI 15 3 0.0135 (0.0135) 0.0068 h x x LWK, ASW, YRI 10 3 0.009(0.009)  0.0045 aj x x PUR, TSI, FIN, 9 4 0.0081 (0.0081) 0.0041 CEU q xx CHS, ASW, JPT, 7 5 0.0063 (0.0063) 0.0032 PUR, CHB Table Footnotes:“x” in a box indicates that the amino acid for the variant form isdifferent from the amino acid at that position in form a, the variantamino acid being shown in “Amino Acid Position & Variation” of the tableand the form a amino acid being shown in the first row of the table;amino acids at all other positions of each variant form are identical tothose found in form a. Amino acid numbering is per the numbering shownfor the pro-form in Table 2 below. ¹Number of individuals in 1000Genomes database found to have the allele; ²Number of unique humanethnic populations in 1000 Genomes database in which the allele wasfound to occur; ³Heterozygous human genotype frequency, ie, cumulativefrequency of all genotypes having one occurrence of the variant alleleand one occurrence of another allele (heterozygous state), eg, acgenotype in 1000 Genomes database; ⁴Homozygous human genotype frequency,ie, cumulative frequency of two occurrences of the variant allele(homozygous state), eg, cc genotype in 1000 Genomes database; and ⁵Totalhuman genotype frequency, ie, total of heterozygous plus homozygoushuman genotype frequencies. ⁶Cumulative human allele frequency of alloccurences of the variant allele in 1000 Genotypes database. Form a′ isidentical to form a with the exception that form a′ has a glycine (G) atposition 620 (see US20120093818 (Amgen, Inc)); form a has E at thisposition. (b) Nucleotide Sequence Variations of Selected Alleles Allelea G C A G A A A Nucleotide Position¹ 1:55505647 1:55505668 1:555238021:55523855 1:55524237 1:55527222 1:55529187 Non-Synonymous NucleotideVariation² T T G A G C G Variant ID³ rs11591147 rs11583680 rs28362261rs28362263 rs562556 rs28362277 rs505151 Variant Corresponding Amino AcidVariation Allele 46L 53V 425S 443T 474V 619P 670G f X c X r X X p X X mX e X X h X X aj X X q X X “X” in a box indicates that a variant allelecomprises the non-synonymous nucleotide variation indicated in the 5throw. Table Footnotes: ¹Notation is chromosome number (all positions areon human chromosome 1):coordinate number (Ensembl release 73 - September2013, Genome assembly: GRCh37 (GCA_000001405.13); ²Nucleotide change(compared to allele a nucleotide shown in first row) giving rise to anamino acid change in the variant form (compared to amino acid of allelea); and ³NCBI dbSNP reference number (NCBI dbSNP Build 138 released onApr. 25, 2013).

TABLE 2 Sequences(a) Human PCSK9 Form a Amino Acid Sequence (SEQ ID NO: 1)-“Pro-form” with Signal Sequence                                            46     53MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQItalics = signal sequence 1-30 Courier = pro peptide 31-152lower case = catalytic domain 153-449UPPER CASE = C-terminal domain 450-692Underlined = residues changed from allele a in other sequences (aa residue number shown)The pro-form is the sequence from amino acid number 31 to (and including)amino acid number 692 of SEQ ID NO: 1.The mature form is the sequence from amino acid number 153 to (and including)amino acid number 692 of SEQ ID NO: 1(b) Human PCSK9 Form a Amino Acid Sequence (SEQ ID NO: 3) - “Mature-form”(Numbering and notation as per SEQ ID NO: I above has been retained)slpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrevdifapgediigassdestcfysqsgtsqaaahvaglaammisaepeltlaelrgriihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGWAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ(c) Human PCSK9 Allele a Nucleotide Sequence (SEQ ID NO: 28)-Encoding “Pro-form” Plus Signal SequenceATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC                                                      R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGAGAGGACGGCGACTACGAGGAGCTGGTGGTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggcaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggctgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                           N4255 AAT to AGT                                   A448T GCC to ACCatccacttctctgccssagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgaccccccscctggtggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC      I474V ATC to GTCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    QG19P CAG to CCG E520G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA             E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGACItalics = nucleotide sequence encoding signal sequence (nucleotides 1-90)Courier = nucleotide sequence encoding pro peptide (nucleotides 91-456)lower case = nucleotide sequence encoding catalytic domain (nucleotides 457-1346)UPPER CASE = nucleotide sequence encoding C-terminal domain (nucleotides 1347-2076)Underlined = allelic variations from allele a in other sequences(aa residue number changes and codon changes shown)The pro-form is encoded by nucleotide sequence from nucleotide 91to (and including) nucleotide 2076.The mature form is encoded by nucleotide sequence from nucleotide457 to (and including) nucleotide 2076

Variant Allele Nucleotide Sequences

Thus,

(i) The nucleotide sequence of allele f is identical to SEQ ID NO: 28except that the nucleotide sequence of allele f comprises a GTC codoninstead of an ATC codon at the position labelled “1474V” in SEQ ID NO:28;(ii) The nucleotide sequence of allele c is identical to SEQ ID NO: 28except that the nucleotide sequence of allele c comprises a GGG codoninstead of an GAG codon at the position labelled “E670G” in SEQ ID NO:28;(iii) The nucleotide sequence of allele r is identical to SEQ ID NO: 28except that the nucleotide sequence of allele r comprises a GTC codoninstead of an ATC codon at the position labelled “I474V” in SEQ ID NO:28; and a GGG codon instead of an GAG codon at the position labelled“E670G” in SEQ ID NO: 28;(iv) The nucleotide sequence of allele p is identical to SEQ ID NO: 28except that the nucleotide sequence of allele p comprises a GTC codoninstead of a GCC codon at the position labelled “A53V” in SEQ ID NO: 28;and a GTC codon instead of an ATC codon at the position labelled “I474V”in SEQ ID NO: 28;(v) The nucleotide sequence of allele m is identical to SEQ ID NO: 28except that the nucleotide sequence of allele m comprises a ACC codoninstead of a GCC codon at the position labelled “A443T” in SEQ ID NO:28;(vi) The nucleotide sequence of allele e is identical to SEQ ID NO: 28except that the nucleotide sequence of allele e comprises a AGT codoninstead of an AAT codon at the position labelled “N425S” in SEQ ID NO:28; and a GTC codon instead of an ATC codon at the position labelled“I474V” in SEQ ID NO: 28;(vii) The nucleotide sequence of allele h is identical to SEQ ID NO: 28except that the nucleotide sequence of allele h comprises a ACC codoninstead of a GCC codon at the position labelled “A443T” in SEQ ID NO:28; and a CCG codon instead of a CAG codon at the position labelled“Q619P” in SEQ ID NO: 28;(viii) The nucleotide sequence of allele aj is identical to SEQ ID NO:28 except that the nucleotide sequence of allele aj comprises a CTTcodon instead of an CGT codon at the position labelled “R46L” in SEQ IDNO: 28; and a GTC codon instead of an ATC codon at the position labelled“I474V” in SEQ ID NO: 28; and(ix) The nucleotide sequence of allele q is identical to SEQ ID NO: 28except that the nucleotide sequence of allele q comprises a GTC codoninstead of a GCC codon at the position labelled “A53V” in SEQ ID NO: 28;and a GGG codon instead of an GAG codon at the position labelled “E670G”in SEQ ID NO: 28.

Variant Pro-Form Amino Acid Sequences (Numbering is as per SEQ ID NO: 1recited above)

(A) The amino acid sequence of form f is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form fcomprises a valine at position 474;(B) The amino acid sequence of form c is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form ccomprises a glycine at position 670;(C) The amino acid sequence of form r is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form rcomprises a valine at position 474 and a glycine at position 670;(D) The amino acid sequence of form p is identical the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form pcomprises a valine at position 53 and a valine at position 474;(E) The amino acid sequence of form m is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form mcomprises a threonine at position 443;(F) The amino acid sequence of form e is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form ecomprises a serine at position 425 and a valine at position 474;(G) The amino acid sequence of form h is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form hcomprises a threonine at position 443 and a proline at position 619;(H) The amino acid sequence of form aj is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form ajcomprises a leucine at position 46 and a valine at position 474; and(I) The amino acid sequence of form q is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form qcomprises a valine at position 53 and a glycine at position 670.

Variant Mature Form Amino Acid Sequences (Numbering is as per SEQ ID NO:1 recited above)

(A′) The amino acid sequence of form f is identical to SEQ ID NO: 2except that the amino acid sequence of form f comprises a valine atposition 474;(B′) The amino acid sequence of form c is identical to SEQ ID NO: 2except that the amino acid sequence of form c comprises a glycine atposition 670;(C′) The amino acid sequence of form r is identical to SEQ ID NO: 2except that the amino acid sequence of form r comprises a valine atposition 474 and a glycine at position 670;(D′) The amino acid sequence of form p is identical to SEQ ID NO: 2except that the amino acid sequence of form p comprises a valine atposition 474;(E′) The amino acid sequence of form m is identical to SEQ ID NO: 2except that the amino acid sequence of form m comprises a threonine atposition 443;(F′) The amino acid sequence of form e is identical to SEQ ID NO: 2except that the amino acid sequence of form e comprises a serine atposition 425 and a valine at position 474;(G′) The amino acid sequence of form h is identical to SEQ ID NO: 2except that the amino acid sequence of form h comprises a threonine atposition 443 and a proline at position 619;(H′) The amino acid sequence of form aj is identical to SEQ ID NO: 2except that the amino acid sequence of form aj comprises valine atposition 474; and(I′) The amino acid sequence of form q is identical to SEQ ID NO: 2except that the amino acid sequence of form q comprises a glycine atposition 670.

The mature form of p is identical to the mature form off and aj.

The mature form of c is identical to the mature form of q.

Further sequence analysis and 3D in silico modelling (see FIG. 1 )revealed that selected variants also fulfilled the following additionalselection criteria:—

-   -   PCSK9 variants whose variant amino acid residues (versus the        common form of human PCSK9) are found in the mature form of the        target (ie, outside the pro-domain); and    -   PCSK9 variants whose variant amino acid residues (versus the        common form of human PCSK9) are surface-exposed on the target,        which the inventor saw as contributing to determining the        topography of the target and potentially contributing to how and        where ligand binding on the target occurs.

As shown in FIG. 1 , identified positions 425, 443, 474, 619 and 670(found in the selected variants of the invention) are allsurface-exposed and outside of the pro-domain. Variant positions 425 and443 are surface-exposed on the catalytic domain, while variant positions474, 619 and 670 are surface-exposed on the C-terminal domain.

The inventor, thus, applied the novel selection criteria to determinerare variant forms of human PCSK9, realising the utility of theirnucleotide and amino acid sequences in the various configurations,aspects, clauses, embodiments and example of the invention herein, andthereby providing for novel personalized medical and diagnosticapplications as described above.

Tailoring Antibodies to Ram PCSK9 Variant Profile

The invention includes the possibility to tailor treatment of humansfurther by selecting antibody-based ligands with variable domains basedon gene segments commonly found in humans of the ethnic populationswhere the variant PCSK9 forms are found to meet the selection criteriaof the invention. An example is provided below for ligands comprisingantibody VH domains derived from recombination of human VH3-23.

The inventor analysed the frequencies and distribution of various humanVH3-23 alleles and realised the desirability of using ligands based onhuman VH3-23 alleles comprising SNP rs56069819. This SNP corresponds toa change from leucine at position 24 in the encoded protein sequence toa valine at that position (L24V change) and the SNP is at coordinate106268889 on human chromosome 14.

FIG. 2 shows the cumulative allele frequency distribution across the1000 Genomes Project database of human VH3-23 alleles comprising SNPrs56069819 (such alleles denoted “C” and the most frequent allele (whichdoes not comprise this SNP) denoted “A”). The figure shows that VH3-23alleles comprising SNP rs56069819 are present at a cumulative frequencyof 11% across all human ethnic populations taken as a whole, whereas incertain specific human ethnic sub-populations (ASW, LWK, YRI, CEU andGBR) such alleles are present at an above-average cumulative frequency.Indicated in the figure are those human PCSK9 variant forms (marked“Variants”) that are found in the various sub-populations withabove-average occurrence of human VH3-23 alleles comprising SNPrs56069819. Table 6 shows the VH3-23 variants and the SNPs that theycomprise, as well as their cumulative allele frequencies as found in the1000 Genomes Project database.

Notably, human VH3-23 alleles comprising SNP rs56069819 were found inthe CEU population at a frequency that is almost double the frequency of11% for all populations. For the ASW and YRI populations the frequencywas over a quarter of the population. Thus, the invention advantageouslyenables one to select a ligand comprising an antibody or antibodyfragment, wherein the antibody or fragment comprises a VH domain derivedfrom the recombination of a human VH gene segment, a human D genesegment and a human JH gene segment, the VH gene segment comprising anucleotide sequence that comprises SNP rs56069819 (dbSNP numbering,build number as recited above).

In an example, one can tailor the treatment further by selecting such aligand that specifically binds to a human PCSK9 selected from forms: f,c, m, e, h, p, q and aj, such forms being those appearing in humanpopulations ASW, LWK, YRI, CEU and GBR.

In an example, the VH gene segment is VH3-23*04, which is a commonlyfound variant that comprises SNP rs56069819 in human populations ASW,LWK, YRI, CEU and GBR.

In an example, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human that expresses a humanPCSK9 selected from forms: f, c, m, e, h, p, q and aj.

In an example, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of ASW, LWK, YRI, CEU orGBR ancestry.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of ASW ancestry, whereinthe human expresses a PCSK9 selected from f, c, m, e, h, p and q or thehuman comprises a corresponding nucleotide or amino acid sequence as setout in Table 5. Optionally this ligand comprises a VH domain derivedfrom recombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of LWK ancestry, whereinthe human expresses a PCSK9 selected from f, c, m, e and h or the humancomprises a corresponding nucleotide or amino acid sequence as set outin Table 5. Optionally this ligand comprises a VH domain derived fromrecombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of YRI ancestry, whereinthe human expresses a PCSK9 selected from f, c, m, e and h or the humancomprises a corresponding nucleotide or amino acid sequence as set outin Table 5. Optionally this ligand comprises a VH domain derived fromrecombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of CEU ancestry, whereinthe human expresses a PCSK9 selected from f, c, p and aj or the humancomprises a corresponding nucleotide or amino acid sequence as set outin Table 5. Optionally this ligand comprises a VH domain derived fromrecombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of GBR ancestry, whereinthe human expresses a PCSK9 selected from f, c, and p or the humancomprises a corresponding nucleotide or amino acid sequence as set outin Table 5. Optionally this ligand comprises a VH domain derived fromrecombination of human VH3-23*04.

In an example, the ligand is alirocumab.

REFERENCES

The references cited herein are incorporated by reference in theirentirety.

-   1) Horton et al, Trends Biochem Sci. 2007 February; 32(2):71-7. Epub    2007 Jan. 9, Molecular biology of PCSK9: its role in LDL metabolism.-   2) Seidah and Prat, J Mol Med (Berl). 2007 July; 85(7):685-96. Epub    2007 Mar. 10, The proprotein convertases are potential targets in    the treatment of dyslipidemia.-   3) Benjannet et al, J Biol Chem. 2004 Nov. 19; 279(47):48865-75.    Epub 2004 Sep. 9, NARC-1/PCSK9 and its natural mutants: zymogen    cleavage and effects on the low density lipoprotein (LDL) receptor    and LDL cholesterol.-   4) Lagace et al, J Clin Invest. 2006 November; 116(11):2995-3005,    Secreted PCSK9 decreases the number of LDL receptors in hepatocytes    and in livers of parabiotic mice.-   5) Maxwell et al, Proc Natl Acad Sci USA. 2005 Feb. 8;    102(6):2069-74. Epub 2005 Jan. 27, Overexpression of PCSK9    accelerates the degradation of the LDLR in a post-endoplasmic    reticulum compartment.-   6) Park et al, J Biol Chem. 2004 Nov. 26; 279(48):50630-8. Epub 2004    Sep. 22, Post-transcriptional regulation of low density lipoprotein    receptor protein by proprotein convertase subtilisin/kexin type 9a    in mouse liver.-   7) Rashid et al, Proc Natl Acad Sci USA. 2005 Apr. 12;    102(15):5374-9. Epub 2005 Apr. 1, Decreased plasma cholesterol and    hypersensitivity to statins in mice lacking Pcsk9.-   8) Kotowski et al, Am J Hum Genet. 2006 March; 78(3):410-22. Epub    2006 Jan. 20, A spectrum of PCSK9 alleles contributes to plasma    levels of low-density lipoprotein cholesterol.-   9) Chen et al, J Am Coll Cardiol. 2005 May 17; 45(10):1611-9. Epub    2005 Apr. 21, A common PCSK9 haplotype, encompassing the E670G    coding single nucleotide polymorphism, is a novel genetic marker for    plasma low-density lipoprotein cholesterol levels and severity of    coronary atherosclerosis.-   10) Pisciotta et al, Atherosclerosis. 2006 June; 186(2):433-40. Epub    2005 Sep. 23, Additive effect of mutations in LDLR and PCSK9 genes    on the phenotype of familial hypercholesterolemia.-   11) Zhao et al, Am J Hum Genet. 2006 September; 79(3):514-23. Epub    2006 Jul. 18, Molecular characterization of loss-of-function    mutations in PCSK9 and identification of a compound heterozygote.-   12) Seidah et al, Proc Natl Acad Sci USA. 2003 Feb. 4;    100(3):928-33. Epub 2003 Jan. 27, The secretory proprotein    convertase neural apoptosis-regulated convertase 1 (NARC-1): liver    regeneration and neuronal differentiation.

TABLE 3 1000 GENOMES PROJECT HUMAN POPULATIONS Below is a summary of theethnic populations as per the 1000 Genomes Project sequences. PopulationEuropean ancestry Utah residents (CEPH) with Northern and WesternEuropean ancestry (CEU) Toscani in Italia (TSI) British from England andScotland (GBR) Finnish from Finland (FIN) Iberian populations in Spain(IBS) East Asian ancestry Han Chinese in Beijing, China (CHB) Japanesein Toyko, Japan (JPT) Han Chinese South (CHS) Chinese Dai inXishuangbanna (CDX) Kinh in Ho Chi Minh City, Vietnam (KHV) Chinese inDenver, Colorado (CHD) (pilot 3 only) West African ancestry Yoruba inIbadan, Nigeria (YRI) Luhya in Webuye, Kenya (LWK) Gambian in WesternDivision, The Gambia (GWD) Malawian in Blantyre, Malawi (MAB) WestAfrican Population (TBD) Americas African Ancestry in Southwest US (ASW)African American in Jackson, MS (AJM) African Caribbean in Barbados(ACB) Mexican Ancestry in Los Angeles, CA (MXL) Puerto Rican in PuertoRico (PUR) Colombian in Medellin, Colombia (CLM) Peruvian in Lima, Peru(PEL) South Asian ancestry Ahom in the State of Assam, India Kayadtha inCalcutta, India Reddy in Hyderabad, India Maratha in Bombay, IndiaPunjabi in Lahore, Pakistan

TABLE 5  PCSK9 SEQUENCES FORM/ SEQ  ALLELE VERSION SEQUENCE ID NO:AMINO ACID SEQUENCES Italics = signal sequence 1-30Courier = pro peptide 31-152 lower case = catalytic domain 153-449UPPER CASE = C-terminal domain 450-692Underlined = residues changed from allele a in other sequences (aa residue number shown)a Pro-Form                                             46     53 withMGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP1 SignalWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSequenceVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ aPro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP 2WRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ aMature formslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshg3thlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ fPro-Form                                             46     53 4 withMGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPSignalWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSequenceVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATA VARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ fPro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP 5WRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATA VARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQf, p, aj  Mature formslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshg6thlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQ cPro-Form                                             46     53 7 withMGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPSignalWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSequenceVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQ cPro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP 8WRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQc, q Mature formslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshg9thlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQ rPro-Form                                             46     53 10 withMGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPSignalWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSequenceVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATA VARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQ rPro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP 11WRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATA VARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQ rMature formslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshg12thlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATA VARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQ pPro-Form                                             46     53 13 withMGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGL VEAPEHGTTATFHRCAKDP SignalWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSequenceVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATA VARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ pPro-Form QEDEDGDYEELVLALRSEEDGL V EAPEHGTTATFHRCAKDP 14WRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATA VARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ mPro-Form                                             46     53 15 withMGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPSignalWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSequenceVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpniva tlppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ 16m Pro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpniva tlppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ mMature formslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshg17thlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpniva tlppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 e Pro-Form                                            46     53 18 withMGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPSignalWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSequenceVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474 dvi seawfpedqrvltpniva t lppsthGAGWQLFCRTVWSAHSGPTRMATA VARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ ePro-Form QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP 19WRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474 dvi seawfpedqrvltpniva t lppsthGAGWQLFCRTVWSAHSGPTRMATA VARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ eMature formslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshg20thlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474 dvi seawfpedqrvltpniva t lppsthGAGWQLFCRTVWSAHSGPTRMATA VARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ hPro-Form                                             46     53 21 withMGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPSignalWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSequenceVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpniva tlppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPPEQVTVACEEGWTLTGCSALPGTSHVLGAY                     670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ h Pro-FormQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDP 22WRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpniva tlppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPPEQVTVACEEGWTLTGCSALPGTSHVLGAY                     670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ h Mature formslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshg23thlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpniva tlppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPPEQVTVACEEGWTLTGCSALPGTSHVLGAY                     670AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ aj Pro-Form                                            46     53 24 withMGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLAL LSEEDGLAEAPEHGTTATFHRCAKDP SignalWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSequenceVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATA VARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ ajPro-Form QEDEDGDYEELVLAL L SEEDGLAEAPEHGTTATFHRCAKDP 25WRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATA VARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ qPro-Form                                             46     53 26 withMGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGL VEAPEHGTTATFHRCAKDP SignalWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHSequenceVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQ qPro-Form QEDEDGDYEELVLALRSEEDGL V EAPEHGTTATFHRCAKDP 27WRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQslpwnlerltppryradeyqppdggslvevylldtslqsdhrelegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevltvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvaglaammlsaepeltlaelrqrlihfsak  425               443                            474dvineawfpedqrvltpnivaalppsthGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT                                            619 620HKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAY                    670 AVDNTCVVRSRDVSTTGSTSE G AVTAVAICCRSRHLAQASQELQNUCLEOTIDE SEQUENCESItalics = nucleotide sequence encoding signal sequence (nucleotides 1-90)Courier = nucleotide sequence encoding pro peptide (nucleotides 91-456)lower case = nucleotide sequence encoding catalytic domain (nucleotides 457-1346)UPPER CASE = nucleotide sequence encoding C-terminal domain (nucleotides 1347-2076)aATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC28                                                       R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGAGAGGACGGCGACTACGAGGAGCTGGTGGTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggcaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggctgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                           N4255 AAT to AGT                                   A448T GCC to ACCatccacttctctgccssagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgaccccccscctggtggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC      I474V ATC to GTC ACAGCCGTCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    QG19P CAG to CCG E520G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA             E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC fATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC29                                                       R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGAGAGGACGGCGACTACGAGGAGCTGGTGGTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggcaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggctgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                           N4255 AAT to AGT                                   A448T GCC to ACCatccacttctctgccssagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgaccccccscctggtggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC      I474V ATC to GTCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    QG19P CAG to CCG E520G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA             E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC cATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC30                                                       R46L CGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGAGAGGACGGCGACTACGAGGAGCTGGTGGTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggcaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggctgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                           N4255 AAT to AGT                                   A448T GCC to ACCatccacttctctgccssagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgaccccccscctggtggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC      I474V ATC to GTCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    QG19P CAG to CCG E520G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA             E670G GAG to GGGCTACAGGCAGCACCAGCGAAGGGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC rATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC31                                                       R46LCGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGAGAGGACGGCGACTACGAGGAGCTGGTGGTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggcaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggctgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                           N4255 AAT to AGT                                   A448T GCC to ACCatccacttctctgccssagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgaccccccscctggtggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC      I474V ATC to GTC ACAGCCGTCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    QG19P CAG to CCG E520G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA             E670G GAG to GGGCTACAGGCAGCACCAGCGAAGGGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC pATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC32                                                       R46LCGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGAGAGGACGGCGACTACGAGGAGCTGGTGGTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGTCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggcaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggctgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                           N4255 AAT to AGT                                   A448T GCC to ACCatccacttctctgccssagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgaccccccscctggtggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC      I474V ATC to GTC ACAGCCGTCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    QG19P CAG to CCG E520G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA             E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC mATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC33                                                       R46LCGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGAGAGGACGGCGACTACGAGGAGCTGGTGGTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggcaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggctgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                           N4255 AAT to AGT                                   A448T GCC to ACCatccacttctctgccssagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgaccccccscctggtggccaccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC      I474V ATC to GTCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    QG19P CAG to CCG E520G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA             E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC eATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC34                                                       R46LCGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGAGAGGACGGCGACTACGAGGAGCTGGTGGTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggcaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggctgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                           N4255 AAT to AGT                                   A448T GCC to ACCatccacttctctgccssagatgtcatcagtgaggcctggttccctgaggaccagcgggtactgaccccccscctggtggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC      I474V ATC to GTCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    QG19P CAG to CCG E520G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA             E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC hATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC35                                                       R46LCGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGAGAGGACGGCGACTACGAGGAGCTGGTGGTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggcaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggctgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                           N4255 AAT to AGT                                   A448T GCC to ACCatccacttctctgccssagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgaccccccscctggtggccaccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC      I474V ATC to GTCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    QG19P CAG to CCG E520G GAG to GGGTCCCGGCCCCTCCGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA             E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC ajATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC36                                                       R46LCGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGAGAGGACGGCGACTACGAGGAGCTGGTGGTAGCCTTGCTTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggcaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggctgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                           N4255 AAT to AGT                                   A448T GCC to ACCatccacttctctgccssagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgaccccccscctggtggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC      I474V ATC to GTC ACAGCCGTCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    QG19P CAG to CCG E520G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA             E670G GAG to GGGCTACAGGCAGCACCAGCGAAGAGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC qATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC37                                                       R46LCGT to CTTCCGCGGGCGCCCGTGCGCAGGAGGAGAGGACGGCGACTACGAGGAGCTGGTGGTAGCCTTGCGTTCCG             A53V GCC to GTCAGGAGGACGGCCTGGTCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGagcatcccgtggaacctggagcggattacccctccacggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggcaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggctgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg                           N4255 AAT to AGT                                   A448T GCC to ACCatccacttctctgccssagatgtcatcaatgaggcctggttccctgaggaccagcgggtactgaccccccscctggtggccgccctgccccccagcacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC      I474V ATC to GTCACAGCCATCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA    QG19P CAG to CCG E520G GAG to GGGTCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA             E670G GAG to GGGCTACAGGCAGCACCAGCGAAGGGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGAC

TABLE 6 Human VH3-23 Variant Alleles VH3-23 Cumulative allele haplotypefrequency SNPs a (=VH3-23*04) 0.0983 rs56069819 d 0.0087 rs56069819 rs61750837  rs61752504 e 0.0046 rs56069819 rs1064090 rs1055799 i 0.0009rs56069819 rs1055799 u 0.0005 rs56069819 rs1064091 s 0.0005 rs56069819rs1064091 rs61752504 rs61750837 r 0.0005 rs56069819 rs1064090 TOTAL:0.114

TABLE 7 Exemplary anti-PCSK9 antibodies and/or antibody fragments usefulin any and all aspects of the invention Patent or patent Publicationwhich is SEQ ID NOs comprising an anti-PCSK9 incorporated by referencein its entirety, and monoclonal antibody or fragment thereofspecifically with respect to the SEQ ID Nos. which are incorporated byreference from the comprising an anti-PCSK9 monoclonal patentapplication publication numbers recited antibody or fragment thereofcited in the in the abutting column. abutting column. Light chaincomplementary determining regions US20120020975 A1 (CDRL) SEQ ID NO: 5,7, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28,30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 270, 271, 272, 273,275, 277, 286, 287, 288, 297, 299, 301, 405, 407, 409, 411, 413, 415,417, 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, 461, 465, 469,473, 477, 481, 485; Heavy chain complementary determining regions (CDRH)SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62,64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91,278, 289, 290, 291, 292, 298, 300, 302, 401, 404, 406, 408, 410, 412,414, 416, 419, 423, 427, 431, 435, 439, 443, 447, 451, 455, 459, 463,467, 471, 475, 479, 483; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16,US20120027765A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33,35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417,465; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60,62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89,91, 404, 406, 408, 410, 412, 414, 416, 463; CDRL SEQ ID NO: 5, 7, 9, 10,12, 13, 15, 16, 17, US8168762B2 18, 19, 20, 21, 22, 23, 24, 26, 28, 30,31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413,415, 417, 465; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83,85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 463; CDRL SEQ ID NO:5, 7, 9, 10, 12, 13, 15, 16, 17, US20120020976A1 18, 19, 20, 21, 22, 23,24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 222,229, 238, 405, 407, 409, 411, 413, 415, 417; CDRH SEQ ID NO: 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74,76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 247, 256, 265, 404, 406,408, 410, 412, 414, 416; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16,17, US20130085265A1 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33,35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417,461, 465, 485; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83,85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 459, 463, 483; CDRLSEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, US20130079501A1 18, 19, 20,21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44,46, 405, 407, 409, 411, 413, 415, 417, 461, 465, 485; CDRH SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69,71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408,410, 412, 414, 416, 459, 463, 483; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13,15, 16, 17, US20120213797A1 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31,32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413,415, 417, 158, 162, 395, 473, 477; CDRH SEQ ID NO: 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77,78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416,180, 175, 308, 368, 471, 475; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15,16, 17, US20120251544A1 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32,33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415,417; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60,62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89,91, 404, 406, 408, 410, 412, 414, 416; CDRL SEQ ID NO: 5, 7, 9, 10, 12,13, 15, 16, 17, US20130052201A1 18, 19, 20, 21, 22, 23, 24, 26, 28, 30,31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413,415, 417, 461, 465, 485; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80,81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 459, 463,483; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, US20130058944A118, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39,40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417, 461, 465, 485; CDRHSEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64,65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404,406, 408, 410, 412, 414, 416, 459, 463, 483; CDRL SEQ ID NO: 5, 7, 9,10, 12, 13, 15, 16, 17, US20130079502A1 18, 19, 20, 21, 22, 23, 24, 26,28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409,411, 413, 415, 417; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81,83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416; CDRL SEQ ID NO:5, 7, 9, 10, 12, 13, 15, 16, 17, US20130245235A1 18, 19, 20, 21, 22, 23,24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405,407, 409, 411, 413, 415, 417; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78,79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416;

1. A method of reducing cholesterol level or maintaining previouslyreduced cholesterol level in a human in need of treatment thereof andwherein said human comprises a VH gene segment selected from the groupconsisting of: (i) a VH3-23 gene segment comprising SNP rs56069819, SNPrs61750837, and SNP rs61752504, and not comprising any of SNP rs1055799,SNP rs1064090 and SNP rs1064091; (ii) a VH3-23 gene segment comprisingSNP rs56069819, SNP rs1064090, and SNP rs1055799, and not comprising anyof SNP rs61750837, SNP rs61752504, and SNP rs1064091; (iii) a VH3-23gene segment comprising SNP rs56069819 and SNP rs1055799, and notcomprising any of SNP rs61750837, SNP rs61752504, SNP rs1064090 and SNPrs1064091; (iv) a VH3-23 gene segment comprising SNP rs56069819 and SNPrs1064091 and not comprising any of SNP rs61750837, SNP rs61752504, SNPrs1055799, and SNP rs1064090; (v) a VH3-23 gene segment comprising SNPrs56069819, SNP rs1064091, SNP rs61752504, and SNP rs61750837, and notcomprising SNP rs1055799, and SNP rs1064090; and (vi) a VH3-23 genesegment comprising SNP rs56069819 and SNP rs1064090, and not comprisingany of SNP rs61750837, SNP rs61752504, SNP rs1055799, and SNP rs1064091.the method comprising administering to said human an antibody orantibody fragment thereof that specifically binds a human proproteinconvertase subtilisin/kexin type 9 (PCSK9), wherein the antibody orantibody fragment comprises the variable domains of alirocumab.
 2. Themethod of claim 1, wherein the PCSK9 comprises an amino acid variationand the human expresses a PCSK9 comprising said amino acid variation. 3.The method of claim 1, wherein the PCSK9 comprises an amino acidvariation selected from the group consisting of I474V and E670G in SEQID NO:
 1. 4. The method of claim 3, wherein the human expresses a PCSK9comprising said selected amino acid variation.
 5. The method of claim 1,wherein the PCSK9 is encoded by the nucleotide sequence of SEQ ID NO: 29or SEQ ID NO:
 30. 6. The method of claim 5, wherein the human expressesa PCSK9 comprising the nucleotide sequence of SEQ ID NO: 29 or SEQ IDNO:
 30. 7. The method of claim 1, wherein the VH gene segment is SEQ IDNO: 31 or
 34. 8. The method of claim 1, wherein the antibody or fragmentcomprises a human gamma-1 heavy chain constant region that comprises anAsp corresponding to position 204 of SEQ ID NO: 42 and a Leucorresponding to position 206 of SEQ ID NO:
 42. 9. The method of claim8, wherein the human comprises an IGHG1*01 human heavy chain constantregion gene segment. 10-14. (canceled)
 15. The method of claim 1,wherein the antibody or fragment is administered by intravenous orsubcutaneous administration and/or is comprised in an injectablepreparation.
 16. (canceled)
 17. The method of claim 3, wherein themethod comprises administering alirocumab to the human.
 18. The methodof claim 4, wherein the method comprises administering alirocumab to thehuman.
 19. The method of claim 5, wherein the method comprisesadministering alirocumab to the human.
 20. The method of claim 6,wherein the method comprises administering alirocumab to the human. 21.The method of claim 7, wherein the method comprises administeringalirocumab to the human.
 22. The method of claim 8, wherein the methodcomprises administering alirocumab to the human.
 23. The method of claim9, wherein the method comprises administering alirocumab to the human.24-28. (canceled)
 29. The method of claim 15, wherein the methodcomprises administering alirocumab to the human.